Anti-aP2 Antibodies and Antigen Binding Agents to Treat Metabolic Disorders

ABSTRACT

This invention is in the area of improved anti-aP2 antibodies and antigen binding agents, and compositions thereof, which target the lipid chaperone aP2/FABP4 (referred to as “aP2”) for use in treating disorders such as diabetes, obesity, cardiovascular disease, fatty liver disease, and/or cancer, among others. In one aspect, improved treatments for aP2 mediated disorders are disclosed in which serum aP2 is targeted and the biological activity of aP2 is neutralized or modulated using low-binding affinity aP2 monoclonal antibodies, providing lower fasting blood glucose levels, improved systemic glucose metabolism, increased systemic insulin sensitivity, reduced fat mass, reduced liver steatosis, reduced cardiovascular disease and/or a reduced risk of developing cardiovascular disease.

RELATED APPLICATIONS

This application is related to and claims the benefit of provisionalU.S. Application No. 62/155,217, filed Apr. 30, 2015, provisional U.S.Application No. 62/232,148, filed Sep. 24, 2015, and provisional U.S.Application No. 62/268,257, filed Dec. 16, 2015. The entirety of theseprovisional applications are hereby incorporated by reference for allpurposes.

FIELD OF THE INVENTION

This invention is in the area of improved anti-aP2 antibodies andantigen binding agents, and compositions thereof, which target the lipidchaperone aP2/FABP4 (referred to as “aP2”) for use in treating disorderssuch as diabetes, obesity, cardiovascular disease, fatty liver disease,and/or cancer, among others. In one aspect, improved treatments for aP2mediated disorders are disclosed in which serum aP2 is targeted and thebiological activity of aP2 is neutralized or modulated using low-bindingaffinity aP2 monoclonal antibodies, providing lower fasting bloodglucose levels, improved systemic glucose metabolism, increased systemicinsulin sensitivity, reduced fat mass, reduced liver steatosis, reducedcardiovascular disease and/or a reduced risk of developingcardiovascular disease.

INCORPORATION BY REFERENCE

The contents of the text file named “15020-001US1SequenceListing_ST25updated.txt” which was created on Apr. 25, 2016 andis 258 KB in size, are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Human adipocyte lipid-binding protein (aP2) belongs to a family ofintracellular lipid-binding proteins involved in the transport andstorage of lipids (Banzszak et al., (1994) Adv. Protein Chem. 45,89-151). The aP2 protein is involved in lipolysis and lipogenesis andhas been indicated in diseases of lipid and energy metabolism such asdiabetes, atherosclerosis, and metabolic syndromes. aP2 has also beenindicated in the integration of metabolic and inflammatory responsesystems. (Ozcan et al., (2006) Science 313(5790):1137-40; Makowski etal., (2005) J Biol Chem. 280(13):12888-95; and Erbay et al., (2009) NatMed. 15(12):1383-91). More recently, aP2 has been shown to bedifferentially expressed in certain soft tissue tumors such as certainliposarcomas (Kashima et al., (2013) Virchows Arch. 462, 465-472).

aP2 is highly expressed in adipocytes and regulated byperoxisome-proliferator-activated receptor-γ (PPARγ) agonists, insulin,and fatty acids (Hertzel et al., (2000) Trends Endocrinol. Metab. 11,175-180; Hunt et al., (1986) PNAS USA 83, 3786-3790; Melki et al.,(1993) J. Lipid Res. 34, 1527-1534; Distel et al., (1992) J. Biol. Chem.267, 5937-5941). Studies in aP2 deficient mice (aP2−/−) indicateprotection against the development of insulin resistance associated withgenetic or diet-induced obesity and improved lipid profile in adiposetissue with increased levels of C16:1n7-palmitoleate, reducedhepatosteatosis, and improved control of hepatic glucose production andperipheral glucose disposal (Hotamisligil et al., (1996) Science 274,1377-1379; Uysal et al., (2000) Endocrinol. 141, 3388-3396; Cao et al.,(2008) Cell 134, 933-944).

In addition, genetic deficiency or pharmacological blockade of aP2reduces both early and advanced atherosclerotic lesions in theapolipoprotein E-deficient (ApoE−/−) mouse model (Furuhashi et al.,(2007) Nature, June 21; 447(7147):959-65; Makowski et al., (2001) NatureMed. 7, 699-705; Layne et al., (2001) FASEB 15, 2733-2735; Boord et al.,(2002) Arteriosclerosis, Thrombosis, and Vas. Bio. 22, 1686-1691).Furthermore, aP2-deficiency leads to a marked protection against earlyand advanced atherosclerosis in apolipoprotein E-deficient (ApoE−/−)mice (Makowski et al., (2001) Nature Med. 7, 699-705; Fu et al., (2000)J. Lipid Res. 41, 2017-2023). Hence, aP2 plays a critical role in manyaspects of development of metabolic disease in preclinical models.

In the past two decades, the biological functions of FABPs in generaland aP2 in particular have primarily been attributed to their action asintracellular proteins. Since the abundance of aP2 protein in theadipocytes is extremely high, accounting for up to few percent of thetotal cellular protein (Cao et al., (2013) Cell Metab. 17(5):768-78),therapeutically targeting aP2 with traditional approaches has beenchallenging, and the promising success obtained in preclinical models(Furuhashi et al., (2007) Nature 447, 959-965; Won et al., (2014) NatureMat. 13, 1157-1164; Cai et al., (2013) Acta Pharm. Sinica 34, 1397-1402;Hoo et al., (2013) J. of Hepat. 58, 358-364) has been slow to progresstoward clinical translation.

In addition to its presence in the cytoplasm, it has recently been shownthat aP2 is actively secreted from adipose tissue through anon-classical regulated pathway (Cao et al., (2013) Cell Metab. 17(5),768-778; Ertunc et al., (2015) J. Lipid Res. 56, 423-424). The secretedform of aP2 acts as a novel adipokine and regulates hepatic glucoseproduction and systemic glucose homeostasis in mice in response tofasting and fasting-related signals. Serum aP2 levels are significantlyelevated in obese mice, and blocking circulating aP2 improves glucosehomeostasis in mice with diet-induced obesity (Cao et al., (2013) CellMetab. 17(5):768-78). Importantly, the same patterns are also observedin human populations where secreted aP2 levels are increased in obesityand strongly correlate with metabolic and cardiovascular diseases inmultiple independent human studies (Xu et al., (2006) Clin. Chem. 53,405-413; Yoo et al., (2011) J. Clin. Endocrin. & Metab. 96, E488-492;von Eynatten et al., (2012) Arteriosclerosis, Thrombosis, and Vas. Bio.32, 2327-2335; Suh et al., (2014) Scandinavian J. Gastro. 49, 979-985;Furuhashi et al., (2011) PloS One 6, e27356; Ishimura et al., (2013)PloS One 8, e81318; Karakas et al., (2009) Metabolism: Clinical andExperimental 58, 1002-1007; Kaess et al., (2012) J. Endocrin. & Metab.97, E1943-1947; Cabre et al., (2007) Atherosclerosis 195, e150-158).Finally, humans carrying a haploinsufficiency allele which results inreduced aP2 expression are protected against diabetes and cardiovasculardisease (Tuncman et al., (2006) PNAS USA 103, 6970-6975; Saksi et al.,(2014) Circulation, Cardiovascular Genetics 7, 588-598).

Cao et al. used a rabbit anti-mouse aP2 polyclonal antibody to show areduction in plasma aP2 levels in obese antibody-treated mice, whichoccurred without any alteration in aP2 protein levels in the adiposetissue (Cao et al., (2013) Cell Metab. 17(5): 768-778; PCT PublicationWO 2010/102171). Administration of the antibody in obese mice did notalter the body weight, but did cause a significant decrease in fastingblood glucose levels within two weeks of treatment compared to controlstreated with a pre-immune IgG. In a glucose tolerance test, micereceiving the aP2 polyclonal antibody exhibited significantly improvedglucose disposal curves compared to control animals.

Miao et al. reported the use of a high affinity mouse anti-human aP2monoclonal antibody (identified as mAb 2E4) to achieve improved high-fatdiet (HFD) induced inflammation in antibody treated mice receiving ahigh-fat diet (Miao et al., (2015) Molecular and Cellular Endocrinology403, 1-9). Treatment with the high affinity mAb 2E4, however, resultedin drastically increased body weights compared with control animals, andno notable change was observed in basal glucose levels after six weeksof treatment. Furthermore, mAb 2E4 treatment failed to affectHFD-induced insulin tolerance.

It is an object of the invention to identify new compounds, methods, andcompositions for the treatment of metabolic disorders.

It is in particular an object of the invention to identify newcompounds, methods, and compositions for the reduction of fasting bloodglucose levels, the improvement of systemic glucose metabolism, theimprovement of glucose tolerance, the increase in systemic insulinsensitivity, the reduction in fat mass, the reduction in fat celllipolysis, the reduction in hepatic glucose production, the reduction inhyperinsulinemia, and/or the reduction in liver steatosis.

It is also an object of the invention to identify new compounds,methods, and compositions for the treatment of diabetes, obesity, anddyslipidemia.

It is further object of the invention to identify new compounds,methods, and compositions for the treatment of inflammatory induceddisorders, for example atherosclerosis.

It is another object of the invention to identify new compounds,methods, and compositions for the treatment of a tumor, cancer, or otherneoplasm.

SUMMARY OF THE INVENTION

Anti-aP2 monoclonal antibodies and antigen binding agents are providedthat have superior and unexpected activity for the treatment ofaP2-mediated disorders. In one embodiment, anti-aP2 monoclonalantibodies and antigen binding agents are provided that contain a lightchain or light chain fragment having a variable region, wherein saidvariable region comprises one, two, or three complementarity determiningregions (CDRs) independently selected from Seq. ID No. 7, Seq. ID No. 8,and Seq. ID No. 9. In another embodiment, anti-aP2 monoclonal antibodiesand antigen binding agents are provided that comprise a light chain orlight chain fragment having a variable region, wherein said variableregion comprises one, two, or three CDRs independently selected fromSeq. ID No. 10, Seq. ID No. 11, Seq. ID No. 12, Seq. ID No. 13, Seq. IDNo. 597, Seq. ID No. 598, or Seq. ID No. 599. In still anotherembodiment, anti-aP2 monoclonal antibodies and antigen binding agentsare provided that comprise a light chain or light chain fragment havinga variable region, wherein said variable region comprises one, two, orthree CDRs independently selected from Seq. ID No. 7, Seq. ID No. 8 andSeq. ID No. 9, Seq. ID No. 10, Seq. ID No. 11, Seq. ID No. 12, Seq. IDNo. 13, Seq. ID No. 597, Seq. ID No. 598, or Seq. ID No. 599. In oneembodiment, anti-aP2 monoclonal antibodies and antigen binding agentsare provided that comprise a light chain or light chain fragment havinga variable region, wherein said variable region comprises Seq. ID No. 7,Seq. ID. No. 8, and at least one CDR selected from Seq. ID. No. 9, Seq.ID No. 10, Seq. ID No. 11, Seq. ID No. 12, Seq. ID No. 13, Seq. ID No.597, Seq. ID No. 598, or Seq. ID No. 599. Alternatively, one or more ofthe disclosed and selected CDRs can be altered by substitution of one ormore amino acids that do not adversely affect or that improve theproperties of the antibody or antigen binding agent, as furtherdescribed herein. In one embodiment, the selected CDR(s) is/are placedin a human immunoglobulin framework. In one embodiment, the humanimmunoglobulin framework is further modified or altered to maintain thebinding affinity specificity of the grafted CDR region.

One of the unexpected discoveries disclosed herein is that the describedantibodies and antigen binding agents do not tightly bind aP2 protein.Typically, antibodies and antigen binding agents are sought that havetight binding affinity (very low KD), as was reported by Miao, et al.(See Background of the Invention). It has been discovered that anantibody or antigen binding agent that binds to aP2 protein in itssecreted (non-cytosolic) state with a weaker binding affinity having aKD of about ≧10⁻⁷ M, has an improved ability to neutralize secreted aP2and cause a significant inhibitory effect on aP2-mediated disorders. Incertain embodiments, the anti-aP2 monoclonal antibody or antigen bindingagent has a KD for human aP2 of between about 10⁻⁴ to 10⁻⁶ M. In otherexamples, the anti-aP2 monoclonal antibody or antigen binding agent hasa KD for human aP2 of about >500 nM, for example, about 500 nM to about10 μM. In another embodiment, the anti-aP2 monoclonal antibody orantigen binding agent has a KD for human aP2 of about 1 μM to about 7μM, or 2 μM to about 5 μM. In an alternative embodiment, the anti-aP2monoclonal antibody has a low binding affinity for mouse aP2 in itsnative, conformational form, for example, in the ranges specified above.

The inventors have also surprisingly found that mice treated with theantibodies described herein maintained total circulating aP2 levels at alevel similar to or slightly lower than that seen in control-treatedanimals. These findings are in contrast to those observed with higheraffinity antibodies, including H3, where treatment of mice with thishigh affinity antibody leads to a dramatic 10-fold increase in totalcirculating aP2 levels. The dramatic increase in aP2 levels seen in micetreated with high affinity antibodies may be due to the increasedhalf-life of the aP2 protein, which generally has a short-half life,when complexed with a high-affinity aP2 antibody.

When administered to a host in need thereof, these anti-aP2 antibodiesand antigen binding agents neutralize the activity of secreted aP2 andprovide lower fasting blood glucose levels, improved systemic glucosemetabolism, increased systemic insulin sensitivity, reduced fat mass,liver steatosis, improved serum lipid profiles, and reduced atherogenicplaque formation in a host when compared to anti-aP2 monoclonalantibodies having higher binding affinities. Therefore, the anti-aP2antibodies and antigen binding agents described herein are particularlyuseful to treat metabolic disorders including, but not limited to,diabetes (both type 1 and type 2), hyperglycemia, obesity, fatty liverdisease, dyslipidemia, polycystic ovary syndrome (POS), a proliferativedisorder such as a tumor or neoplasm, (including, but not limited to,for example, transitional bladder cancer, ovarian cancer, andliposarcoma), atherosclerosis, and other cardiovascular disorders byadministering an effective amount to a host, typically a human, in needthereof.

Without wishing to be bound by any one theory, it is believed thatvarious tissues contribute to circulating aP2 levels. For example, it isbelieved that adipose tissue contributes to levels of circulating aP2.In addition, it is believed that other tissues, for example macrophages,contribute to circulating levels of aP2. In one embodiment, a host isadministered an anti-aP2 antibody or antigen binding agent describedherein to treat an aP2 mediated disorder. In one embodiment, a host isadministered an anti-aP2 antibody or antigen binding agent describedherein to treat an aP2 mediated disorder wherein the disorder ismediated by adipose tissue-contributed circulating aP2. In oneembodiment, a host is administered an anti-aP2 antibody or antigenbinding agent described herein to treat an aP2 mediated disorder whereinthe disorder is mediate by macrophage-contributed circulating aP2.

In one embodiment, the anti-aP2 antibody or antigen binding agentincludes at least one CDR selected from Seq. ID Nos. 7-13 or Seq. IDNos. 597-599, and at least one CDR selected from CDRH1 (Seq. ID NO. 14),CDRH1 variant 1 (Seq. ID No. 15), CDRH1 variant 2 (Seq. ID No. 600),CDRH2 (Seq. ID No. 16), CDRH2 variant 1 (Seq. ID No. 17), CDRH2 variant2 (Seq. ID No. 18), CDRH2 variant 3 (Seq. ID No. 601), CDHR3 (Seq. IDNo. 19), CDHR3 variant 1 (Seq. ID No. 20), CDRH3 variant 2 (Seq. ID No.21), or CDRH3 variant 3 (Seq. ID No. 602), wherein the CDR sequences aregrafted into a human immunoglobulin framework. In one embodiment, thehuman immunoglobulin framework is further modified or altered tomaintain the binding affinity specificity of the grafted CDR region.

In certain embodiments, the anti-aP2 antibody or antigen binding agentincludes at least the light chain variable sequence 909 gL1 (Seq. ID No.446), the light chain sequence 909 gL1 VL+CL (Seq. ID No. 447), thelight chain variable sequence 909 gL10 (Seq. ID No. 448), the lightchain sequence 909 gL10 VL+CL (Seq. ID No. 449), the light chainvariable sequence 909 gL13 (Seq. ID No. 487), the light chain sequence909 gL13 VL+CL (Seq. ID No. 489), the light chain variable sequence 909gL50 (Seq. ID No. 488), the light chain sequence 909 gL50 VL+CL (Seq. IDNo. 490), the light chain variable sequence 909 gL54 (Seq. ID No. 450),the light chain sequence 909 gL54 VL+CL (Seq. ID No. 451), the lightchain variable sequence 909 gL55 (Seq. ID No. 452) or the light chainsequence 909 gL55 VL+CL (Seq. ID No. 453).

In other embodiments, the anti-aP2 antibody or antigen binding agentincludes a light chain variable sequence selected from 909 gL1 (Seq. IDNo. 446), 909 gL10 (Seq. ID No. 448), 909 gL13 (Seq. ID No. 487), 909gL50 (Seq. ID No. 488), 909 gL54 (Seq. ID No. 450), or 909 gL55 (Seq. IDNo. 452), and a heavy chain variable sequence selected from 909 gH1(Seq. ID No. 455), 909 gH14 (Seq. ID No. 457), 909 gH15 (Seq. ID No.459), 909 gH61 (Seq. ID No. 461), and 909 gH62 (Seq. ID No. 463). Forexample, the antibody or antigen binding agent can include at least thelight chain variable sequence 909 gL1 (Seq. ID No. 446) and the heavychain variable sequence 909 gH1 (Seq. ID. No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL10 (Seq. ID No. 448) and the heavychain variable sequence 909 gH1 (Seq. ID No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL10 (Seq. ID No. 448) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL1 (Seq. ID No. 446) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL13 (Seq. ID No. 487) and the heavychain variable sequence 909 gH1 (Seq. ID No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL13 (Seq. ID No. 487) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL50 (Seq. ID No. 488) and the heavychain variable sequence 909 gH1 (Seq. ID No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL50 (Seq. ID No. 488) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL54 (Seq. ID No. 450) and the heavychain variable sequence 909 gH1 (Seq. ID No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL54 (Seq. ID No. 450) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL55 (Seq. ID No. 452) and the heavychain variable sequence 909 gH1 (Seq. ID No. 455). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL55 (Seq. ID No. 452) and the heavychain variable sequence 909 gH15 (Seq. ID No. 459). In one embodiment,the anti-aP2 antibody or antigen binding agent can include at least thelight chain variable sequence 909 gL1 (Seq. ID No. 446) and the heavychain variable sequence 909 gH14 (Seq. ID. No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL10 (Seq. ID No. 448) and the heavychain variable sequence 909 gH14 (Seq. ID No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL13 (Seq. ID No. 487) and the heavychain variable sequence 909 gH4 (Seq. ID No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL50 (Seq. ID No. 488) and the heavychain variable sequence 909 gH4 (Seq. ID No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL54 (Seq. ID No. 450) and the heavychain variable sequence 909 gH14 (Seq. ID No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL55 (Seq. ID No. 452) and the heavychain variable sequence 909 gH14 (Seq. ID No. 457). In one embodiment,the anti-aP2 antibody or antigen binding agent can include at least thelight chain variable sequence 909 gL1 (Seq. ID No. 446) and the heavychain variable sequence 909 gH61 (Seq. ID. No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL10 (Seq. ID No. 448) and the heavychain variable sequence 909 gH61 (Seq. ID No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL13 (Seq. ID No. 487) and the heavychain variable sequence 909 gH61 (Seq. ID No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL50 (Seq. ID No. 488) and the heavychain variable sequence 909 gH61 (Seq. ID No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL54 (Seq. ID No. 450) and the heavychain variable sequence 909 gH61 (Seq. ID No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL55 (Seq. ID No. 452) and the heavychain variable sequence 909 gH61 (Seq. ID No. 461). In one embodiment,the anti-aP2 antibody or antigen binding agent can include at least thelight chain variable sequence 909 gL1 (Seq. ID No. 446) and the heavychain variable sequence 909 gH62 (Seq. ID. No. 463). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL10 (Seq. ID No. 448) and the heavychain variable sequence 909 gH62 (Seq. ID No. 463). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL13 (Seq. ID No. 487) and the heavychain variable sequence 909 gH62 (Seq. ID No. 463). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL50 (Seq. ID No. 488) and the heavychain variable sequence 909 gH62 (Seq. ID No. 463). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL54 (Seq. ID No. 450) and the heavychain variable sequence 909 gH62 (Seq. ID No. 463). In one embodiment,the anti-aP2 antibody or antigen binding agent includes at least thelight chain variable sequence 909 gL55 (Seq. ID No. 452) and the heavychain variable sequence 909 gH62 (Seq. ID No. 463). The anti-aP2monoclonal antibodies, and where relevant the antigen binding agents,described herein containing the variable light and/or variable heavychain sequences containing the CDRs described herein may furthercomprise constant region domains selected having regard to the proposedfunction of the antibody molecule, and in particular the effectorfunctions which may be required. For example, the constant regiondomains may be human IgA, IgD, IgE, IgG, or IgM domains. In particular,human IgG constant region domains may be used, especially for examplethe IgG1 and IgG3 isotypes when the antibody molecule is intended fortherapeutic uses and antibody effector functions are required.Alternatively, IgG2 and IgG4 isotypes may be used when the antibodymolecule is intended for therapeutic purposes and antibody effectorfunctions are not required. It will be appreciated that sequencevariants of these constant region domains may also be used. For exampleIgG4 molecules in which the serine at position 241 (IgG4P) has beenchanged to proline as described in Angal et al., Molecular Immunology,1993, 30 (1), 105-108 may be used, and are contemplated herein.

In one embodiment, the anti-aP2 antibody comprises a light chainvariable sequence Rabbit Ab 909 VL region (Seq. ID No. 445), and furtheroptionally comprises a heavy chain variable sequence Rabbit Ab 909 VHregion (Seq. ID No. 454).

In one embodiment, a low binding affinity monoclonal anti-aP2 antibodyCA33, a rabbit-mouse hybrid anti-aP2 monoclonal antibody, which includesRabbit 909 VH (Seq. ID No. 454) and 909 VL (Seq. ID No. 445), isdescribed that lowers fasting blood glucose levels, improves systemicglucose metabolism, increases systemic insulin sensitivity and reducesfat mass and liver steatosis in obese mice.

It has been found that CA33 binds to both lipid-bound and lipid-free aP2at similar affinities (See FIGS. 2H and 2I, respectively). These datasuggest that the efficacy of CA33 is not mediated by its binding onlyapo-aP2 or only aP2 molecules that carry a specific lipid. It has alsobeen found that the CA33 epitope does not overlap with the hinge region(which contains E15, N16, and F17) and it does not appear that CA33binding alters ligand access to the hydrophobic pocket of aP2. In fact,at neutral pH, paranaric acid binding to aP2 is similar in the presenceor absence of CA33, supporting the conclusion that antibody binding toaP2 does not block overall lipid binding (See FIG. 2G).

Furthermore, it has been discovered that exogenous aP2 treatment leadsto the disassociation of a novel transcriptional holocomplex composed ofForkhead box protein O1 (FoxO1) and the transcriptional corepressorC-terminal binding protein 2 (CtBP2) in hepatocytes, leading toexpression of gluconeogenic genes. In vivo, the FoxO1/CtBP2 interactionis readily detectable in the liver of lean mice, but markedly decreasedin the context of obesity, a setting in which the level of circulatingaP2 is markedly increased. It is shown herein that administration ofrecombinant aP2 decreases the FoxO1/CtBP2 interaction while theinteraction increases in the setting of aP2 genetic deficiency andantibody-mediated neutralization. It has further been shown herein thatCtBP2 overexpression in the liver of obese mice dramatically amelioratesglucose intolerance as well as hepatic steatosis through repression ofgluconeogenic and lipogenic gene expression. In one embodiment of theinvention, an anti-aP2 antibody is administered for the treatment of adisorder in a host, including a human, associated with the misregulationof the FoxO1/CtBP2 pathway. In one embodiment, improved treatments forFoxO1-mediated disorders or CtBP2-mediated disorders are disclosed inwhich serum aP2 is targeted and the biological activity of aP2 isneutralized or modulated using a low-binding affinity aP2 monoclonalantibody described herein, wherein the expression level of one or moreFoxO1-regulated or CtBP2-regulated genes is reduced. In one embodiment,provided herein is a method of modulating the expression of a FoxO1and/or CtBP2-regulated gene comprising administering to a host alow-binding affinity aP2 monoclonal antibody described herein. See Jacket al. “C-terminal binding protein: A metabolic sensor implicated inregulating adipogenesis.” Int J Biochem Cell Biol. 2011 May;43(5):693-6; Vernochet C, et al. “C/EBPalpha and the corepressors CtBP1and CtBP2 regulate repression of select visceral white adipose genesduring induction of the brown phenotype in white adipocytes byperoxisome proliferator-activated receptor gamma agonists.” Mol CellBiol. 2009 September; 29(17):4714-28; Kajimura, S. et al. “Regulation ofthe brown and white fat gene programs through a PRDM16/CtBPtranscriptional complex.” Genes Dev. 2008 May 15; 22 (10): 1397-409.

Antigen binding agents may be in any form that provides the desiredresults. As non-limiting examples, the form of the binding agent mayinclude a single chain fragment, Fab fragment, Fab′ fragment, F(ab′)2fragment, a scFv, a scAb, single domain light chain, a single domainheavy chain, a synthetic antigen binding agent that includes a naturallyoccurring or non-naturally occurring linking moiety between two or morefragments (for example a compound that links two or more of the lightchain CDRs described herein or a variant thereof with one or more aminoacid substitutions), an antigen binding agent conjugated for targeteddelivery, as well as any peptide obtained from or derived from such anantibody.

In one aspect, the present invention provides a polynucleotide, such asDNA, encoding an antibody or fragment as described herein, for exampleas provided in Table 12. Also provided is a host cell comprising saidpolynucleotide.

Specifically, the invention includes administering an effective amountof an anti-aP2 antibody described herein, or a pharmaceuticallyacceptable composition thereof, capable of reducing the activity ofsecreted aP2 (i.e., extracellular aP2) in a body fluid of a host, forexample blood or serum, which results in the attenuation of the severityof, for example, aP2 mediated disorders, including but not limited to ametabolic, cardiovascular, inflammatory, liver, or neoplastic disorderor symptom.

In one aspect of the invention, the purified anti-aP2 monoclonalantibody or antigen binding agent binds to human aP2 protein (Seq. ID.No. 1) with a unique pattern of contact points within 3-4 Angstroms.

In one embodiment, the anti-aP2 monoclonal antibody binds human aP2having the amino acid sequence:

(Seq. ID No. 1) MCDAFVGTWK LVSSENFDDY MKEVGVGFAT RKVAGMAKPN MIISVNGDVITIKSESTFKN TEISFILGQE FDEVTADDRK VKSTITLDGG VLVHVQKWDG KSTTIKRKREDDKLVVECVM KGVTSTRVYE RA ,or a naturally occurring variant thereof. In an alternative embodiment,the anti-aP2 monoclonal antibody binds to a human aP2 protein having anamino acid sequence that is at least 95%, 96%, 97%, 98%, or 99%identical to Seq. ID No. 1. In one embodiment, the anti-aP2 monoclonalantibody binds to a human aP2 protein having an amino acid sequence thathas one or more (for example 1, 2, 3 or 4) amino acid substitutions,additions and/or deletions as compared to Seq. ID No. 1.

In one embodiment, the anti-aP2 monoclonal antibody or antigen bindingagent binds to an epitope selected from an amino acid sequenceunderlined in Seq. ID No. 1 above. In one embodiment, the anti-aP2monoclonal antibody directly interacts with one or more, for example 1,2, 3, 4, 5, 6, 7, 8, or 9, amino acids bolded in Seq. ID No. 1 above. Inone example, the anti-aP2 monoclonal antibody or antigen binding agentbinds to an epitope of the human aP2 protein comprising at least one,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, of amino acids 9-17,amino acids 20-28, or amino acids 118-132 of Seq. ID No. 1, andoptionally has a KD of at least about ≧10⁻⁷ M.

In another example, the anti-aP2 monoclonal antibody or antigen bindingagent thereof binds an epitope of human aP2 comprising one or more, forexample 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more, amino acid residuesselected from 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, 132A (bolded inSeq. ID No. 1, above), or an amino acid residue within about 4 angstromsof any of 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, and 132A, optionallywith a KD for secreted aP2 of about ≧10⁻⁷ M.

In one embodiment, the light chain of the antibody binds an epitope ofhuman aP2 comprising one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, or9 or more, amino acid residues selected from 10K, 11L, 12V, 13S, 37A,38K, 57T, 130E, or 132A, or an amino acid residue within about 4angstroms thereof. In one embodiment, the light chain of the antibodybinds an epitope of human aP2 comprising one or more, for example 1, 2,3, 4, 5, 6, 7, 8, or 9 or more, amino acid residues selected from 10K,11L, 12V, 13S, 37A, 38K, 57T, 130E, or 132A, or an amino acid residuewithin about 4 angstroms thereof, and has a KD of at least about ≧10⁻⁷M.

In one embodiment, the anti-aP2 monoclonal antibody or antigen bindingagent binds to aP2 only through, or primarily through, light chain CDRs.In an alternative embodiment, the anti-aP2 monoclonal antibody orantigen binding agent has light chain CDRs that bind to aP2 with agreater affinity than its heavy chain CDRs bind to aP2. As one example,the antibody or antigen binding agent specifically binds aP2, and doesnot specifically bind to FABP5/Mal1.

Methods of producing the disclosed anti-aP2 antibodies and antigenbinding agents are provided herein as well as methods of conjugating theantibody or fragment to a polymer, such as PEG.

The present disclosure also includes pharmaceutical compositionscomprising an effective amount of one of the anti-aP2 antibodies and/orantigen binding agents in combination with a pharmaceutically acceptablecarrier. The anti-aP2 monoclonal antibody or antigen binding agent canbe administered to the host by any desired route, including intravenous,systemic, topical transdermal, sublingual, buccal, oral, intra-aortal,topical, intranasal, intraocular, or via inhalation. In one embodiment,the anti-aP2 monoclonal antibody or antigen binding agent isadministered to the host via controlled release delivery.

A method of preventing or attenuating the severity of an aP2 mediateddisorder in a host, such as a human, is presented that includesadministering an effective amount of a humanized antibody, for example,an anti-aP2 monoclonal antibody or antigen binding agent describedherein, resulting in the reduction or attenuation of the biologicalactivity of secreted aP2. Nonlimiting examples of uses of the describedanti-aP2 antibodies and antigen binding agents by administering aneffective amount to a host in need thereof include one or a combinationof:

-   -   (i) Reduction of total cholesterol;    -   (ii) Reduction of high density lipoprotein (HDL), low density        lipoprotein (LDL), very low density lipoprotein (VLDL), and/or        triglycerides;    -   (iii) Reduction of fasting blood glucose levels;    -   (iv) Reduction of fat mass levels;    -   (v) Reduction of hepatic glucose production;    -   (vi) Reduction of fat cell lipolysis;    -   (vii) Reduction of hyperinsulinemia;    -   (viii) Reduction of liver steatosis;    -   (ix) Improvement in glucose metabolism;    -   (x) Increase in insulin sensitivity; and/or,    -   (xi) Preventing islet β-cell death, dysfunction, or loss.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-8 are further discussed in Example 1 below.

FIG. 1A is a table listing the binding affinities (KD(M)) of anti-aP2monoclonal antibodies (CA33, CA13, CA15, CA23, and H3) to human andmouse aP2 as determined by biomolecular interaction analysis, using aBiacore T200 system.

FIG. 1B is a schematic describing the in vivo study design and antibodyregimen for the treatment of obese mice (fed a high-fat diet (HFD)) withvehicle or anti-aP2 antibodies. Mice (21 weeks old) were fed HFD for 15weeks prior to antibody administration (n=10 for each group). Anti-aP2antibodies were administered by subcutaneous injection at aconcentration of 33 mg/kg, twice per week.

FIG. 1C is a bar graph showing plasma insulin levels (ng/ml) at week 0(open bars) or week 4 (solid bars) in mice treated with vehicle oranti-aP2 monoclonal antibodies CA33, CA13, CA15, CA23, or H3. * p<0.05.

FIG. 1D is bar graph showing blood glucose levels (mg/dl) at week 0(open bars) or week 4 (solid bars) in obese mice on a high-fat diet(HFD) treated with vehicle or anti-aP2 monoclonal antibodies CA33, CA13,CA15, CA23, or H3. Blood glucose levels were measured after 6 hours ofday-time food withdrawal. * p<0.05, ** p<0.01.

FIG. 1E is a line graph showing glucose levels (mg/dl) vs. time(minutes) during a glucose tolerance test (GTT). The test was performedafter 2 weeks of treatment in obese mice on HFD with vehicle (diamonds)or anti-aP2 monoclonal antibodies (0.75 g/kg glucose)(CA33;squares)(CA15: triangles). * p<0.05.

FIG. 1F is a line graph showing insulin levels (mg/dl) vs. time(minutes) during an insulin tolerance test (ITT). This test wasperformed after 3 weeks of treatment in obese mice on HFD with vehicle(diamonds) or anti-aP2 monoclonal antibodies (0.75 IU/kg insulin) (CA33;squares) (CA15; triangles). ** p<0.01.

FIG. 1G is a bar graph showing body weight (g) at week 0 (open bars) orweek 4 (solid bars) in mice treated with vehicle or anti-aP2 monoclonalantibodies CA33, CA13, CA15, CA23, or H3. Weight was measured in the fedstate. * p<0.05.

FIG. 2A is a bar graph of the signal interaction (nm) as determined byoctet analysis for the anti-aP2 antibodies CA33 and H3 against aP2(black bars) compared to the related proteins FABP3 (gray bars) andFABP5/Mal1 (light gray bars).

FIG. 2B is a bar graph of plasma aP2 levels (ng/ml) as determined byELISA in HFD-fed mice treated with vehicle, CA33, or H3 for 3 weeks(n=10 mice per group). Mice had been on HFD for 12 weeks before theexperiment was initiated. A Western blot to detect aP2 in serum fromthree mice from each group (vehicle, CA33, or H3) is shown in the inset.** p<0.01.

FIG. 2C is a table of antibody crossblocking of H3 vs. CA33, CA13, CA15,and CA23 as determined by Biacore analysis. ++=complete blocking;+=partial blocking; −=no crossblocking.

FIG. 2D shows the epitope sequence of aP2 residues involved in theinteraction with CA33 and H3, as identified by hydrogen-deuteriumexchange mass spectrometry (HDX). Interacting residues are underlined.

FIG. 2E is a superimposed image of the Fab of CA33 co-crystallized withaP2 and the Fab of H3 co-crystallized with aP2.

FIG. 2F is a high resolution mapping of CA33 epitope on aP2. Interactingresidues in both molecules are indicated. Hydrogen bonds are shown asdashed lines. The side chain of K10 in aP2 forms a hydrophobicinteraction with the phenyl side chain of Y92.

FIG. 2G is a line graph showing paranaric acid binding to aP2 (relativefluorescence) vs. pH in the presence of IgG control antibody (circles)or CA33 antibody (squares).

FIG. 2H is a graph showing CA33 binding to aP2 (resonance units) vs.time (seconds) in response to increasing concentrations of lipid-loadedaP2.

FIG. 2I is a graph showing CA33 binding to aP2 (resonance units) vs.time (seconds) in response to increasing concentrations of de-lipidatedaP2.

FIG. 3A is a bar graph showing fasting blood glucose (mg/dl) inHFD-induced obese aP2−/− mice before (open bars) and after CA33 antibodyor vehicle treatment for three weeks (solid bars).

FIG. 3B is a bar graph showing body weight (g) in HFD-induced obeseaP2−/− mice before (open bars) and after CA33 antibody or vehicletreatment for three weeks (solid bars).

FIG. 3C is a line graph showing glucose levels (mg/dl) in HFD-inducedobese aP2−/− mice vs. time (minutes) during a glucose tolerance test(GTT). The test was performed after 2 weeks of vehicle (triangles) orCA33 antibody treatment (squares) in aP2−/− mice.

FIG. 3D is a bar graph showing body weight (g) in ob/ob mice before(open bars) and after (solid bars) 3 weeks of CA33 antibody or vehicletreatment (n=10 mice per group). ** p<0.01.

FIG. 3E is a bar graph showing fasting blood glucose levels (mg/dl) inob/ob mice before (open bars) and after (solid bars) 3 weeks of CA33antibody or vehicle treatment (n=10 mice per group). ** p<0.01.

FIG. 3F is a bar graph showing plasma insulin levels (ng/ml) in ob/obmice following three weeks of vehicle (open bar) or CA33 antibodytreatment (solid bar). ** p<0.01.

FIG. 3G is a line graph showing glucose levels (mg/dl) in ob/ob mice vs.time (minutes) during a glucose tolerance test (GTT). The test wasperformed after 2 weeks of vehicle (triangles) or CA33 antibodytreatment (squares) in aP2−/− mice. * p<0.05.

FIG. 4A is a representative image of hematoxylin and eosin (H&E) stainedliver from HFD-induced obese mice after 5 weeks of treatment withvehicle or CA33. Scale bar is 50 μm.

FIG. 4B is a bar graph of liver triglyceride (TG) content (mg/g oftissue) in HFD-induced obese mice after 5 weeks of treatment withvehicle (open bar) or CA33 antibody (solid bar). * p<0.05.

FIG. 4C is bar graph showing mRNA expression of lipogenic genesstearoyl-CoA desaturase (Scd1), fatty acid synthase (Fasn) andacetyl-CoA carboxylase (Acc1) in liver samples from HFD-induced obesemice after 5 weeks of vehicle (black bars) or CA33 treatment (graybars). ** p<0.01.

FIG. 4D is a bar graph showing levels of plasma nonesterified fatty acid(NEFA) (mg/ml) in HFD-induced obese mice after 5 weeks of vehicle (openbar) or CA33 treatment (solid bar). ** p<0.01.

FIG. 4E is a bar graph showing levels of plasma glycerol (mg/ml) inHFD-induced obese mice after 5 weeks of vehicle (open bar) or CA33treatment (solid bar). * p<0.05.

FIG. 4F is a bar graph showing levels of plasma total cholesterol(mg/dl) in HFD-induced obese mice after 5 weeks of vehicle (open bar) orCA33 treatment (solid bar).* p<0.05.

FIG. 4G is a bar graph showing levels of plasma triglycerides (mg/dl) inHFD-induced obese mice after 5 weeks of vehicle (open bar) or CA33treatment (solid bar).

FIG. 4H is a bar graph showing levels of plasma FABP3 or FABP5 (Mal1)(ng/ml) in HFD-induced obese mice after 5 weeks of vehicle (open bar) orCA33 treatment (solid bar).

FIG. 4I is a bar graph showing levels of plasma glucagon (pg/ml) inHFD-induced obese mice after 5 weeks of vehicle (open bar) or CA33treatment (solid bar).

FIG. 4J is a bar graph showing levels of plasma adiponectin (μg/ml) inHFD-induced obese mice after 5 weeks of vehicle (open bar) or CA33treatment (solid bar).

FIG. 5A is a bar graph of body fat mass (g) (open bar) and lean mass (g)(gray bar) as determined by dual-energy X-ray absorptiometry (DEXA)after 5 weeks of vehicle or CA33 treatment. (n=10 per group). * p<0.05.

FIG. 5B is a bar graph showing liver weight (g) and % body weight ofobese mice after 5 weeks of vehicle (open bars) or CA33 treatment (solidbars). ** p<0.01.

FIG. 5C is a bar graph showing physical activity (activity units—AU) formice in the light or in the dark after 5 weeks of vehicle (open bars) orCA33 treatment (solid bars).

FIG. 5D is a bar graph showing total food intake (g) in obese mice after5 weeks of vehicle (open bar) or CA33-treated mice (solid bar) on HFD(n=8 per group).

FIG. 5E is a bar graph showing VO2 concentrations by volume during thelight and dark periods in mice treated with CA33 (solid bar) for eightweeks compared to vehicle (open bar).

FIG. 5F is a bar graph showing calculated Respiratory Exchange Ratio(RER) during light and dark periods in mice treated with CA33 (solidbar) for eight weeks compared to vehicle (open bar).

FIG. 5G is a bar graph showing the weight of brown adipose tissue (BAT)in mice treated with CA33 (solid bar) for eight weeks compared tovehicle (open bar).

FIG. 5H shows representative H&E stained sections of BAT in micefollowing treatment with CA33 (solid bar) for eight weeks compared tovehicle (open bar).

FIG. 5I is a bar graph showing the weight of perigonadal white adiposetissue (PGWAT) in obese mice after 5 weeks of vehicle (open bar) orCA33-treated mice (solid bar). ** p<0.01.

FIG. 5J is a representative image of hematoxylin and eosin (H&E) stainedepididymal adipose tissue after 5 weeks of treatment with vehicle (leftimage) or CA33 (right image). Scale bar is 200 μm.

FIG. 5K is a bar graph showing F4/80+ Mac cells (%) in adipose tissuedetermined by FACS after 5 weeks of vehicle (open bar) or CA33-treatedmice (solid bar).

FIG. 5L is a bar graph showing CD11b+ cells (%) in adipose tissuedetermined by FACS analysis after 5 weeks of vehicle (open bar) orCA33-treated mice (solid bar).

FIG. 5M is a bar graph showing mRNA levels (mRNA/Tbp) for TNF, IL-1β,IL-6, CCL2, CXCL1, F4/80 or CD68 in perigonadal white adipose tissue(PG-WAT) after 5 weeks of vehicle (open bar) or CA33-treated mice (solidbar).

FIG. 5N is a Western blot showing adipose tissue aP2/FABP4 proteinlevels in mice treated with vehicle or CA33 for 3 weeks. Adipose tissuesamples from aP2−/−, mal1−/−, and aP2/mal1−/− animals were included asprotein controls. β-tubulin is shown as a loading control.

FIG. 5O is a Western blot showing adipose tissue Mal1/FABP5 proteinlevels in mice treated with vehicle or CA33 for 3 weeks. Adipose tissuesamples from aP2−/−, mal1−/−, and aP2/mal1−/− animals were included asprotein controls. β-tubulin is shown as a loading control.

FIG. 5P is bar graph of relative protein levels for either aP2 or mal1in mice treated with vehicle or CA33 for 3 weeks. The results shown inFIG. 5P quantify the Western blots shown in FIGS. 5N and 5O.

FIG. 6A is a bar graph showing mRNA expression of gluconeogenic genesphosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase(G6pc). Liver samples were collected after 6 hours of day-time foodwithdrawal from obese mice treated with vehicle (open bars) or CA33(solid bars) (n=10 for each group) for 4 weeks. * p<0.05.

FIG. 6B is a bar graph showing enzymatic activity of Pck1 (nM/min/g) inliver samples. Liver samples were collected after 6 hours of day-timefood withdrawal from obese mice treated with vehicle (open bars) or CA33(solid bars) (n=10 for each group) for 4 weeks. * p<0.05.

FIG. 6C is a bar graph showing enzymatic activity ofglucose-6-phosphatase (G6pc) (U/mg) in liver microsomal fraction formice treated with vehicle (open bars) or CA33 (solid bars) (n=10 foreach group) for four weeks. * p<0.05.

FIG. 6D is a line graph showing blood glucose (mg/dl) vs. time duringhyperinsulinemic-euglycemic clamp. Clamp studies were performed in obesemice on a high-fat diet (HFD) after five weeks of treatment with vehicle(diamonds) or CA33 (squares) (n=7 for each group).

FIG. 6E is a bar graph showing glucose infusion rate (GIR) (mg/kg/min)in obese mice on a high-fat diet (HFD) after five weeks of treatmentwith vehicle (open bar) or CA33 (solid bar). * p<0.05.

FIG. 6F is a bar graph showing clamp hepatic glucose production (c-HGP)(mg/kg/min) in obese mice on a high-fat diet (HFD) after five weeks oftreatment with vehicle (open bar) or CA33 (solid bar).* p<0.05.

FIG. 6G is a bar graph showing the rate of whole body glucosedisappearance (RD) (mg/kg/min) in obese mice on a high-fat diet (HFD)after five weeks of treatment with vehicle (open bar) or CA33 (solidbar). * p<0.05.

FIG. 6H is a bar graph showing glucose uptake in triceps surae muscle(mg/kg/min) in obese mice on HFD after five weeks of treatment withvehicle (open bar) or CA33 (solid bar). * p<0.05.

FIG. 6I is a bar graph showing whole body glycolysis in obese mice on ahigh-fat diet (HFD) after five weeks of treatment with vehicle (openbar) or CA33 (solid bar). * p<0.05.

FIG. 7A is a line graph showing glucose levels (mg/dl) vs. time(minutes) in a glucose tolerance test (GTT) following two weeks ofselective antibody treatment using high affinity antibodies (CA13, CA15,CA23, and H3) versus vehicle control.

FIG. 7B is a line graph showing glucose levels (mg/dl) vs. time(minutes) in an insulin tolerance test (ITT) following three weeks ofselective antibody treatment using high affinity antibodies (CA13, CA15,CA23, and H3) versus vehicle control.

FIG. 8A is a bar graph showing basal hepatic glucose production(mg/kg/min) in vehicle control (open bar) or CA33 (solid bar) treatedmice during hyperinsulinemic-euglycemic clamp of HFD-mice.

FIG. 8B is a bar graph showing serum insulin levels (ng/ml) in CA33 mice(solid bars) or vehicle control mice (open bars) duringhyperinsulinemic-euglycemic clamp of HFD-mice. ** p<0.01.

FIG. 8C is a bar graph showing glucose uptake (mg/kg/min) in gonadalwhite adipose tissue (GWAT) in vehicle control (open bar) or CA33treated (solid bar) mice during hyperinsulinemic-euglycemic clamp ofHFD-mice.

FIGS. 9-16 are further discussed in Example 2 below.

FIG. 9 is a line graph illustrating diabetes incidence (%) vs. time(weeks of treatment) in the NOD mouse model for Type 1 Diabetes. NODmice were treated with vehicle (diamonds) or the aP2 monoclonal antibodyCA33 (squares).

FIG. 10A is a line graph illustrating death rate (%) vs. time (weeks oftreatment) in the NOD mouse model for Type 1 Diabetes. NOD mice weretreated with vehicle (diamonds) or the aP2 monoclonal antibody CA33(squares).

FIG. 10B is a bar graph illustrating blood glucose level (mg/dL) inPBS-treated or aP2-antibody treated NOD mice following a 6 hr fast.

FIG. 10C is a line graph illustrating insulin level (ng/mL) inPBS-treated or aP2-antibody treated NOD mice following a 6 hr fast.

FIGS. 11A and 11B: NOD aP2^(+/+) and NOD aP2^(−/−) mice were subjectedto glucose tolerance test (GTT) (FIG. 11A) and insulin tolerance test(ITT) (FIG. 11B).

FIG. 12A is a bar graph showing insulin (ng/ml/ug DNA) secretion fromNOD aP2^(+/+) and NOD aP2^(−/−) mice islets after stimulation witheither low or high glucose.

FIGS. 12B and 12C: illustrates bar graphs showing total insulin(ng/ml/ug DNA) content from isolated islets of four (FIG. 12B) and seven(FIG. 12C) week old NOD aP2^(+/+) (left bar) and NOD aP2^(−/−) mice(right bar).

FIG. 13 illustrates the number of islets visible in NOD aP2^(−/−) micecompared to NOD aP2^(+/+) mice following pancreatic dissection.

FIGS. 14A and 14B: illustrates stained beta cells (FIG. 14A), which weresubsequently quantified (FIG. 14B) in NOD aP2^(+/+) and NOD aP2^(−/−)mice.

FIGS. 15A-15C: illustrates bar graphs showing glucose-stimulated insulin(ng/ml/ug DNA) secretion from either a rat insulinoma beta cell line(INS1) (FIG. 15A), aP2-deficient c57b/6 mice (Mouse Islets) (FIG. 15B),or human islets (FIG. 15C) after stimulation with either low or highglucose.

FIG. 15D illustrates that aP2 is taken up into mouse islets after 20minutes of treatment with 10 ug/ml aP2 (n=5).

FIGS. 15E and 15F: bar graphs showing insulin (ng/ml/ug DNA) secretionfollowing aP2 treatment for 24 hrs under “fasting” conditions fromeither INS1 beta cells (FIG. 15E) or primary islets isolated fromaP2^(−/−) mice (FIG. 15F) after stimulation with either low or highglucose.

FIG. 16 is a diagram of an inducible model of Type 1 diabetes in mice(rat insulin promoter-lymphocytic choriomeningitis virus-glycoprotein,or RIP-LCMV-GP, mice). Mice are injected with LCMV, which leads todestruction of β-cells and the development of diabetes.

FIG. 17 is a schematic showing an aP2-antibody administration schedulein a Type 1 diabetes-induced mouse model (RIP-LCMV-GP mice) in a groupof aP2−/− (Group C) and aP2-normal mice injected twice weekly witheither 33 mg/kg of CA33 (Group B) or PBS control (Group A) and fed anormal chow diet.

FIG. 18 is a line graph showing 6-hour fasting blood glucosemeasurements (mg/dl) vs. time (days) in a Type 1 diabetes-induced mousemodel (RIP-LCMV-GP mice) in a group of aP2−/− (triangles) and aP2-normalmice injected twice weekly with either 33 mg/kg of CA33 (squares) orcontrol (PBS) (circles) and fed a normal chow diet. CA33 treated and aP2deficient animals had significantly lower fasting blood glucose afterLCMV administration compared to vehicle treated animals. * p<0.05, **p<0.01, *** p<0.005.

FIG. 19 is a bar graph showing incidence of Type 1 diabetes in a Type 1diabetes-induced mouse model (RIP-LCMV-GP mice) in a group of aP2−/−(triangles) and aP2-normal mice injected twice weekly with either 33mg/kg of CA33 (squares) or control (PBS) (circles) and fed a high fatdiet. Diabetes was defined as a 6-hour fasting blood glucose measurementgreater than 250 mg/dl. CA33 treatment provided protection againstdevelopment of type 1 diabetes in the RIP-LCMV-GP model similar to thatobserved in aP2 genetically deficient animals.

FIG. 20A is a bar graph showing islet infiltration (%; non-insulitis(open bars); peri-insulitis (checkered bars); mild insulitis (graybars); severe insulitis (black bars)) in GP+ control mice, GP+ aP2−/−mice, or GP+ mice treated with CA33, 14 days after injection with LCMV.Mice were treated with 1.5 mg CA33 or vehicle by injection twice weekly,starting 14 days prior to LCMV infection. Insulitis scoring wasperformed on H&E stained pancreatic sections. Each islet was scored aseither “non-insulitis,” “peri-insulitis,” “mild insulitis” or “severeinsulitis.” The bar graph represents the percentage of each islet typein each animal analyzed.

FIG. 20B is a representative picture of an islet showing little to noinsulitis.

FIG. 20C is a representative picture of an islet showing severeinsulitis.

FIG. 20D is a bar graph showing islets with mild insulitis (%) in GP+control mice, GP+aP2−/− mice, or GP+ mice treated with CA33, 14 daysafter injection with LCMV. Mice were treated with 1.5 mg CA33 or vehicleby injection twice weekly, starting 14 days prior to LCMV infection. *p<0.05; ** p<0.01.

FIG. 20E is a bar graph showing islets with severe insulitis (%) in GP+control mice, GP+ aP2−/− mice, or GP+ mice treated with CA33, 14 daysafter injection with LCMV. Mice were treated with 1.5 mg CA33 or vehicleby injection twice weekly, starting 14 days prior to LCMV infection. **p<0.01; *** p<0.005.

FIG. 21A is a series of representative images of islets stained for ATF6(left column) or XBP1 (right column) in GP+ control mice, GP+ aP2−/−mice, or GP+ mice treated with CA33, 14 days after injection with LCMV.Mice were treated with 1.5 mg CA33 or vehicle by injection twice weekly,starting 14 days prior to LCMV infection.

FIG. 21B is a bar graph showing ATF6 levels (relative fluorescenceintensity, RFI) in pancreatic samples from GP+ control mice, GP+ aP2−/−mice, or GP+ mice treated with CA33, 14 days after injection with LCMV.Mice were treated with 1.5 mg CA33 or vehicle by injection twice weekly,starting 14 days prior to LCMV infection. ** p<0.01; *** p<0.005.

FIG. 21C is a bar graph showing sXBP1 levels (relative fluorescenceintensity, RFI) in pancreatic samples from GP+ control mice, GP+ aP2−/−mice, or GP+ mice treated with CA33, 14 days after injection with LCMV.Mice were treated with 1.5 mg CA33 or vehicle by injection twice weekly,starting 14 days prior to LCMV infection. ** p<0.01; *** p<0.005.

FIGS. 22-26 are further discussed in Example 3 below.

FIG. 22A is table providing the antibody dose (μg) and injection volume(μl) calculations to achieve a 33 mg/kg dosage based on average bodyweight of the ApoE knockout mice (atherosclerosis mouse model) at theindicated time points (weeks).

FIG. 22B is a graph showing atherosclerotic lesion area (%) in ApoEknockout mice treated with PBS (circles), CA33 (squares), or CA15(triangles). Aortas from sacrificed ApoE knockout mice were dissectedfrom the proximal aorta to the iliac bifurcation, and the aortae werepinned out in an en face preparation. En face pinned-out aortas werestained with Sudan IV. Quantitation of lesion areas was achieved usingImageJ software developed at the NIH. The outer perimeter of the pinnedout aorta was defined in the software to establish the total area of theaorta as a white background. The percent area of the lesions stained redwith Sudan IV was then measured and calculated by the software. *p<0.05.

FIG. 22C is a representative image of an en face pinned aorta from anApoE knockout mouse fed western diet and treated with vehicle for twelveweeks.

FIG. 22D is a representative image of an en face pinned aorta from anApoE knockout mouse fed western diet and treated with CA33 (33 mg/kg)for twelve weeks.

FIG. 22E is a representative image of an en face pinned aorta from anApoE knockout mouse fed western diet and treated with CA15 (33 mg/kg)for twelve weeks.

FIG. 22F is a line graph showing average body weight (g) of ApoEknockout mice vs. age (weeks old) in mice treated for twelve weeks withPBS (circles), CA33 (squares), or CA15 (triangles). ApoE knockout micewere fed western diet and treated with vehicle or antibody (33 mg/kg)for twelve weeks.

FIG. 22G illustrates aP2 protein expression in adipose tissue ofaP2^(adip−/−) mice.

FIGS. 22H-22J: are bar graphs showing the level of aP2 (ng/ml) (FIG.22H), triglyceride (mg/dl) (FIG. 22I), and cholesterol (mg/dl) (FIG.22J) in ApoE^(−/−)aP2^(adip+/+) and ApoE^(−/−)aP2^(adip−/−) mice after12 weeks of western diet.

FIG. 22K is a bar graph showing the atherosclerotic lesion area inApoE^(−/−)aP2^(adip+/+) and ApoE^(−/−)aP2^(adip−/−) mice.

FIG. 22L is a representative image of an en face pinned aorta fromApoE^(−/−)aP2^(adip+/+) and ApoE^(−/−)aP2^(adip−/−) mice fed westerndiet for 12 weeks.

FIG. 23A is a bar graph showing average body weight (g) of ApoE knockoutmice treated for twelve weeks with PBS (black bar), CA33 (light graybar), or CA15 (gray bar). ApoE knockout mice were fed western diet andtreated with vehicle or antibody (33 mg/kg) for twelve weeks.CA33-treated mice show a statistically significant lower average bodyweight than vehicle treated mice. * p<0.05.

FIG. 23B is a bar graph showing liver weight (g) of ApoE knockout micetreated for twelve weeks with PBS (black bar), CA33 (light gray bar), orCA15 (gray bar). ApoE knockout mice were fed western diet and treatedwith vehicle or antibody (33 mg/kg) for twelve weeks. CA33 treated miceshow a statistically significant lower average liver weight than vehicletreated mice. * p<0.05.

FIG. 23C is a bar graph showing body weight minus liver weight (g) ofApoE knockout mice treated for twelve weeks with PBS (black bar), CA33(light gray bar), or CA15 (gray bar). ApoE knockout mice were fedwestern diet and treated with vehicle or antibody (33 mg/kg) for twelveweeks. CA33 treated mice show a lower average body weight minus liverweight than vehicle treated mice.

FIG. 23D is a bar graph showing average body weight, lean mass, or fatmass (g) of ApoE knockout mice treated for twelve weeks with PBS (blackbar), CA33 (light gray bar), or CA15 (gray bar). ApoE knockout mice werefed western diet and treated with vehicle or antibody (33 mg/kg) fortwelve weeks and body weight, lean mass, and fat mass were measured bydual X-ray absorbance (DEXA) spectroscopy.

FIG. 24A is a bar graph of fasting basal glucose levels (mg/dl) prior toa glucose tolerance test in ApoE knockout mice treated with PBS (blackbar), CA33 (light gray bar), or CA15 (gray bar). ApoE knockout mice werefed western diet and treated with vehicle or antibody (33 mg/kg) fortwelve weeks. CA33-treated mice have statistically significant lowerfasting blood glucose than vehicle treated mice. * p<0.05.

FIG. 24B is a line graph of glucose levels (mg/dl) vs. time (minutes)during a glucose tolerance test in ApoE knockout mice treated with PBS(triangles), CA33 (squares), or CA15 (circles). ApoE knockout mice werefed western diet and treated with vehicle or antibody (33 mg/kg) fortwelve weeks. The glucose tolerance test was performed by oral glucoseadministration (1.0 g/kg) on conscious mice after an overnight (16 h)fast.

FIG. 25A is a bar graph of cholesterol in lipoprotein fractions (mg/dl)(total lipoprotein, very low-density lipoprotein (VLDL), low-densitylipoprotein (LDL), or high-density lipoprotein (HDL)) in ApoE knockoutmice treated with PBS (triangles), CA33 (squares), or CA15 (circles) forsix weeks. Particle size distribution of the lipoproteins was determinedby fast-performance liquid chromatography (FPLC), using pooled samplesof plasma. * p<0.05.

FIG. 25B is a bar graph of cholesterol in lipoprotein fractions (mg/dl)(total lipoprotein, very low-density lipoprotein (VLDL), low-densitylipoprotein (LDL), or high-density lipoprotein (HDL)) in ApoE knockoutmice treated with PBS (triangles), CA33 (squares), or CA15 (circles) fortwelve weeks. Particle size distribution of the lipoproteins wasdetermined by fast-performance liquid chromatography (FPLC), usingpooled samples of plasma. * p<0.05.

FIG. 26A is a bar graph of triglycerides in lipoprotein fractions(mg/dl) (total lipoprotein, very low-density lipoprotein (VLDL),low-density lipoprotein (LDL), or high-density lipoprotein (HDL)) inApoE knockout mice treated with PBS (triangles), CA33 (squares), or CA15(circles) for six weeks. Particle size distribution of the lipoproteinswas determined by fast-performance liquid chromatography (FPLC), usingpooled samples of plasma. * p<0.05.

FIG. 26B is a bar graph of triglycerides in lipoprotein fractions(mg/dl) (total lipoprotein, very low-density lipoprotein (VLDL),low-density lipoprotein (LDL), or high-density lipoprotein (HDL)) inApoE knockout mice treated with PBS (triangles), CA33 (squares), or CA15(circles) for twelve weeks. Particle size distribution of thelipoproteins was determined by fast-performance liquid chromatography(FPLC), using pooled samples of plasma. * p<0.05.

FIGS. 27-28 are further discussed in Example 4 below.

FIG. 27 provides anti-human aP2 humanized kappa light chain variableregion antibody fragments, wherein the 909 sequence is rabbit variablelight chain sequence, and the 909 gL1, gL10, gL13, gL50, gL54, and gL55sequences are humanized grafts of 909 variable light chain usingIGKV1-17 human germline as the acceptor framework. The CDRs are shown inbold/underlined, while the applicable donor residues are shown inbold/italic and are highlighted: 2V, 3V, 63K and 70D. The mutation inCDRL3 to remove a Cysteine residue is shown in bold/underlined and ishighlighted: 90A.

FIG. 28 provides anti-human aP2 humanized heavy chain variable regionantibody fragments, wherein the 909 sequence is rabbit variable heavychain sequence, and the 909gH1, gH14, gH15, gH61, and gH62 sequences arehumanized grafts of 909 variable heavy chain using IGHV4-4 humangermline as the acceptor framework. The CDRs are shown inbold/underlined. The two residue gap in framework 3, in the loop betweenbeta sheet strands D and E, is highlighted in gH1: 75 and 76. Applicabledonor residues are shown in bold/italic and are highlighted: 23T, 67F,71K, 72A, 73S, 74T, 77T, 78V, 79D, 89T, and 91F. The mutation in CDRH2to remove a Cysteine residue is shown in bold/underlined and ishighlighted: 59S. The mutation in CDRH3 to remove a potential Aspartateisomerization site is shown in bold/underlined and is highlighted: 98E.The N-terminal Glutamine residue is replaced with Glutamic acid, and isshown in bold and highlighted: 1E.

FIGS. 29-31 are further discussed in Example 5 below.

FIG. 29 is an illustration representing key contact points between aP2and a murine derived anti-aP2 antibody. As is shown in the figure,substitution of particular amino acid residues with CDR regions of theheavy and light chains resulted in a reduction in the affinity of thealtered antibodies in comparison with the parent antibody H3.

FIG. 30A provides amino acid substitutions for inducing a reduction inbinding affinity in a murine derived anti-aP2 antibody compared to theparent antibody H3, where g1=wild-type parent H3. Binding affinities(μM) are shown for CA33 and two reduced affinity H3-like antibodies.

FIG. 30B provides the protein sequence of the heavy chain detailing theamino acid substitutions for inducing a reduction in binding affinity ina murine derived anti-aP2 antibody compared to the parent antibody H3,where g1=wild-type parent H3. The CDRs are underlined and the amino acidsubstitutions are shown in bold.

FIG. 30C provides the protein sequence of the light chain detailing theamino acid substitutions for inducing a reduction in binding affinity ina murine derived anti-aP2 antibody compared to the parent antibody H3,where g1=wild-type parent H3. The CDRs are underlined and the amino acidsubstitutions are shown in bold.

FIG. 31 is a table indicating affinity data against human and mouse aP2for murine derived anti-aP2 antibodies that have been mutated to reducebinding affinity. Binding affinities (KD) are shown for two clones foreach antibody (APP5168/PB1172 and APP5169/PB1171) and average bindingaffinity (KD) is shown (μM).

FIGS. 32-46 are further discussed in Example 6 below.

FIG. 32 illustrates that aP2 regulates gluconeogenic gene expression ina FoxO1-dependent manner. Primary hepatocytes were incubated with orwithout 50 μg/ml of aP2 in the presence or absence of 2 μM forskolin(fsk) for 3 h (FIGS. 32A, 32B, 32C, 32D, and 32E) or 6 h (FIG. 32F).FIGS. 32A and 32B show the impact of forskolin stimulation on the effectof aP2 on gluconeogenic gene expression (G6pc and Pck1 respectively) inhepatocytes, as well as the role of aP2 lipid binding in this process(n=4). mut: lipid binding mutant, delip: delipidated aP2, WT:lipid-loaded aP2. FIG. 32C illustrates the screening of transcriptionfactors by siRNA mediated knockdown and the effect on forskolin andaP2-stimulated G6pc expression (n=4). FIG. 32D illustrates the effect ofadenovirus-mediated FoxO1 knockdown on aP2-mediated G6pc expression(n=4). FIG. 32E illustrates the knockdown efficiency at protein levelsfor FoxO1. FIG. 32F illustrates the effect of aP2 stimulation of FHREluciferase activity in primary hepatocytes (n=6). FIG. 32G illustratesglucose production from hepatocytes following adenovirus-mediated FoxO1knockdown and aP2 stimulation (n=5). Data are expressed as the mean±SEM.*, ** and NS denote p<0.05, p<0.01 and no significant difference,respectively, determined by Student's t-test.

FIG. 33 illustrates that aP2 alters fatty acid metabolism to regulategluconeogenic gene expression. FIGS. 33A and 33B show the tabulatedresults for G6pc and Pck1 expression, respectively, from primaryhepatocytes preincubated with vehicle or 50 μM Etomoxir for 30 min andstimulated with 50 μg/ml of aP2 in the presence or absence of 2 μM offorskolin (fsk) for 3 h (n=4). FIGS. 33C and 33D show the resultsobtained when primary hepatocytes were stimulated with 50 μg/ml of aP2for 2 h, and stimulated with 150 μM palmitate (FIG. 33C) or 150 μMoleate (FIG. 33D) for the indicated amount of time to measure oxygenconsumption rate (OCR, FIG. 33C, n=6) or fatty acid uptake (FIG. 33D,n=5). FIGS. 33E and 33F illustrate the mRNA levels obtained for G6pc andPck1, respectively, when primary hepatocytes were stimulated with 50μg/ml of aP2 for 2 h and the cells were further stimulated with 50 μg/mlof aP2 in the presence or absence of 2 μM of forskolin (fsk)/100 μMpalmitate for 3 h (n=4). FIGS. 33G and 33H illustrate the mRNA levelsobtained for G6pc and Pck1, respectively, when primary hepatocytes werepreincubated with 5 μM Triacsin C for 30 min and further treated as inFIG. 33D with or without the same concentrations of Triacsin C (n=4).FIG. 33I illustrates the level of nuclear fatty acyl-CoA (pmol/10⁶cells) in primary hepatocytes after they were treated with eithervehicle or aP2. Data are expressed as the mean±SEM. *, ** and NS denotep<0.05, p<0.01 and no significant difference, respectively, determinedby Student's t-test.

FIG. 34 demonstrates the existence of a novel FoxO1/CtBP2transcriptional complex regulated by aP2. Primary hepatocytes werestimulated with 50 μg/ml of aP2 (FIGS. 34 A, B, H, L, M and N) in thepresence or absence of 2 μM of forskolin (fsk) for 3 h (FIGS. 34 A andB) or 90 min (FIGS. 34 H, K, M and N). FIGS. 34A and 34B show theeffects of adenovirus-mediated CtBP1 and CtBP2 knockdown on aP2-inducedexpression of G6pc and Pck1, respectively (n=4). FIG. 34C shows thePxDLS-like motifs in human and mouse FoxO sequences as indicated by therectangles. FIGS. 34D and 34E show endogenous FoxO1/CtBP complex inprimary hepatocytes shown by reciprocal coimmunoprecipitation of CtBP orFoxO1, respectively. FIGS. 34F and 34G illustrate the critical role ofthe PxDL motif in the FoxO1/CtBP2 complex. HEK293 cells were transfectedwith either control plasmid, FLAG wild-type FoxO1 or mutant FLAG-FoxO1(Mut: PSDL>PSAS, Δpsdl: PxDL motif deletion) along with CtBP2 expressionplasmid. The complexes were immunoprecipitated with FLAG agarose beads.FIG. 34H shows the dissociation of native FoxO1/CtBP2 complex by aP2treatment. The endogenous FoxO1/CtBP2 complex was immunoprecipitatedfrom primary hepatocytes. FIGS. 34I and 34J show the nutrient sensingcapability of FoxO1/CtBP2 complex. HEK293 cells were transfected withFLAG wild-type FoxO1 and CtBP2. Increasing concentrations of eitheroleoyl-CoA (0, 50, 150, 500 μM) (FIG. 34I) or NADH (0, 10, 30, 100 μM)(FIG. 34J) were added to the cell lysates and immunoprecipitated withFLAG agarose beads. FIG. 34K shows the effect of cytosolic redox statuson FoxO1/CtBP2 complex. HEK293 cells were transfected with FLAGwild-type FoxO1 and CtBP2 and incubated with different ratios of lactateand pyruvate for 1 h. FIG. 34L illustrates the effect of forskolin andaP2 treatment on the nuclear/cytoplasmic localization of FoxO1 andCtBP2. NE: nuclear extract. FIGS. 34M and 34N show chromatinimmunoprecipitation (ChIP) analysis (n=4) of FoxO1 or CtBP2,respectively, at the G6pc promoter. Data are expressed as themean±SEM. * and ** denote p<0.05 and p<0.01, respectively, determined byStudent's t-test.

FIG. 35 shows the fine-tuned regulation of gluconeogenic gene expressionby CtBP2. Primary hepatocytes were stimulated with vehicle or 2 μM offorskolin (fsk) for 3 h (FIGS. 35 A, D and E) or 6 h (B, F) afterknockdown (A, B) or overexpression (C, D, E, F) of CtBP2. FIG. 35A showsthe effect of CtBP2 and/or FoxO1 knockdown on G6pc expression (n=5). They-axis scale is expanded in the inset to show the data in the absence offsk more clearly. FIG. 35B shows FHRE luciferase activity followingCtBP2 knockdown (n=8). FIG. 35C shows GUS and CtBP2 overexpression inprimary hepatocytes. FIGS. 35D and 35E show the regulation ofgluconeogenic gene expression by CtBP2 overexpression (n=4). FIG. 35Fshows FHRE luciferase activity following CtBP2 overexpression (n=8).FIG. 35G shows the effect of acute activation of insulin and cAMPsignaling on the FoxO1/CtBP2 complex. HEK293 cells transfected withFLAG-FoxO1 and CtBP2 were stimulated with either vehicle, 100 nM insulin(ins) or 50 μM of forskolin (fsk) for 30 min.

FIG. 36 shows the regulation of the endogenous FoxO1/CtBP2 complex invivo. FIGS. 36A, 36C, 36E, 36G, and 36J: liver homogenates from thefollowing groups of mice were subjected to co-immunoprecipitaton toassay the endogenous FoxO1/CtBP2 complex. All mice were sacrificed after4-6 h fasting. The densitometric quantification is shown to the right ofeach blot; see FIGS. 36B, 36D, 36F, 36H and 36K. FIGS. 36A and 36B:genetically obese ob/ob mice and their control lean mice. FIGS. 36C and36D: diet-induced obese mice (high fat diet (HFD) for 16 weeks) andtheir control lean mice (normal chow (NC)). FIGS. 36E and 36F: aP2knockout (KO) or their control wild-type (WT) mice on normal chow (NC)or high fat diet (HFD) for 16 weeks. FIGS. 36G and 36H: diet-inducedobese mice (HFD) treated with monoclonal aP2 antibody (CA33) or vehicle,and control lean mice (NC). FIGS. 361, 36J, 36K, 36L, and 36M:recombinant aP2 was administered into wild-type lean mice for 5 days.Serum aP2 levels (FIG. 36I) and FoxO1/CtBP2 complex (FIGS. 36J and 36K),gene expression in liver (FIGS. 36L and 36M) were analyzed (n=4). FIG.36N shows the results from adenovirus-mediated knockdown of CtBP2 in theliver of wild-type lean mice 5 days after transduction (n=6). * and **denote p<0.05 and p<0.01, respectively, determined by Student's t-test.

FIG. 37: CtBP2 gain of function in liver improves glucose tolerance andameliorates steatosis in obese mice. CtBP2 was overexpressed in theliver of diet-induced obese mice (14 weeks on the diet) by adenoviraltransduction. HFD: high fat diet, NC: normal chow. FIGS. 37A and 37Btabulate the measurement of body weights and blood glucose levels,respectively, after overnight fasting (n=4-5). FIGS. 37C, 37D, and 37Q:Dietary induced obese mice transduced with AdGUS or AdCtBP2 weresubjected to glucose tolerance test (GTT, FIG. 37C), insulin tolerancetest (ITT, FIG. 37D), and pyruvate tolerance test (PTT, FIG. 37Q)(n=10-12). FIGS. 37E, 37F and 37G: Gene expression in liver (n=4-5) forG6pc, Pck1, or Alb mRNA, respectively. FIG. 37H illustratesrepresentative hematoxylin and eosin stained sections of liver. FIG. 37Ishows tabulated liver triglyceride content (n=10). FIG. 37J showstabulated serum ALT levels (n=10). FIGS. 37K, 37L, 37M, 37N, and 37Oshow Mlxipl/Srebf1c expression (FIGS. 37K and 37L) and lipogenic geneexpression (FIGS. 37M, 37N and 37O) in the liver (n=10). Mice weresacrificed after overnight fasting. FIG. 37P is a schematic diagramshowing a potential regulatory mechanism. ACSL; acyl-CoA synthetase.Data are expressed as the mean±SEM. *, ** and NS denote p<0.05, p<0.01and no significant difference, respectively, determined by Student'st-test.

FIG. 38: cAMP stimuli amplify aP2-induced gluconeogenic gene expression.FIGS. 38A and 38B show the G6pc mRNA and Pck1 mRNA levels, respectively,from when primary hepatocytes were stimulated with or without 50 μg/mlof aP2 in the absence or presence of 2 μM forskolin (fsk) for 3 h (n=4).The y-axis scale is expanded in the inset to show the data in theabsence of fsk more clearly. In FIGS. 38C and 38D, primary hepatocyteswere treated in the same way as in FIG. 38A with or without PKAinhibitor (H89, 20 μM) (n=4); G6pc mRNA and Pck1 mRNA levels weremeasured and tabulated. Data are expressed as the mean±SEM. ** denotesp<0.01 determined by Student's t-test.

FIG. 39 shows the screening of transcription factor(s) responsible foraP2-mediated upregulation of gluconeogenic genes. FIGS. 39A, 39B and 39Cshow the knockdown efficiency at the mRNA level, corresponding to theexperiment shown in FIG. 32C (n=4). FIGS. 39D, 39E, 39F and 39G show thescreening of the transcription factor(s) responsible for aP2-dependentupregulation of gluconeogenic genes. Primary hepatocytes were treated inthe same way as FIG. 32A after knockdown of Hif1a, Ppargc1a or Stat3(n=4). FIG. 39H shows Foxo1 expression levels as in FIG. 32 (n=4). Dataare expressed as the mean±SEM. * and ** denote p<0.05 and p<0.01,respectively, determined by Student's t-test.

FIG. 40: CtBP2-dependent transcriptional activation by aP2. FIGS. 40A,40B, 40C and 40D show the knockdown efficiency at mRNA and proteinlevels corresponding to FIG. 34 (n=4). FIGS. 40E, 40F and 40G show thegluconeogenic gene expression in primary hepatocytes after adenoviralmediated knockdown of HNF4α (n=4). Gene expression profiles for G6pc andPck1 are shown in FIGS. 40E and 40F, respectively, and knockdownefficiency at protein levels is shown in FIG. 40G. Data are expressed asthe mean±SEM. * and ** denote p<0.05 and p<0.01, respectively,determined by Student's t-test.

FIG. 41: Characterization of the FoxO1/CtBP complex. FIGS. 41A and 41Bshow the results of mutagenesis studies for FoxO1/CtBP1 interaction.HEK293 cells transfected with wild-type FLAG FoxO1 (WT) or mutant FoxO1(Mut: PSDL>PSAS, Δpsdl: PxDL motif deletion) along with CtBP1 expressionplasmid were lysed and subjected to co-immunoprecipitation. FIG. 41Cshows the results of incubating primary hepatocytes with 50 μg/ml aP2 inthe presence or absence of forskolin (fsk, 2 μM) for 2 h and the nativeFoxO1/CtBP2 complex was immunoprecipitated. FIG. 41D shows the cellularlactate/pyruvate ratio. Primary hepatocytes were treated as in FIG. 33E.FIG. 41E shows immunocytochemistry corresponding to FIG. 34L. FIG. 41Fshows levels of phosphorylation and acetylation of FoxO1. Primaryhepatocytes were treated with forskolin (fsk, 2 μM) and/or 50 μg/ml aP2for 30 min.

FIG. 42 shows the effect of CtBP2 overexpression on the expression ofparticular genes. Primary hepatocytes were treated as in FIG. 35D andthe gene expression profile was analyzed.

FIG. 43 illustrates the results of the in vivo administration ofrecombinant aP2. Recombinant aP2 was administered into wild-type leanmice for 5 days (n=4). FIGS. 43A, 43B, 43C, 43D and 43E show results forbody weights before (day 0) and after intraperitoneal injections (day 5)of aP2, serum levels of insulin, glucagon, glycerol, and free fattyacids (FFA), respectively. FIGS. 43F and 43G show the mRNA levels forPck1 and Foxo1, respectively, in wild-type or liver specific FoxO1 KOmice treated with recombinant aP2 (n=7-8). Data are expressed as themean±SEM. NS denotes no significant difference determined by Student'st-test.

FIG. 44: CtBP2 overexpression in vivo. FIG. 44A illustrates the proteinlevels of overexpression of GUS and CtBP2 in liver. FIG. 44B illustratesthe blood glucose levels without normalization to the baseline in theITT study (FIG. 37D, n=10-12). FIG. 44C shows the serum insulin levelsin GUS or CtBP2 overexpressed liver. Data are expressed as themean±SEM. * and ** denote p<0.05 and p<0.01, respectively, determined byStudent's t-test.

FIG. 45: Involvement of CtBP2 in the pathogenesis of hepatic steatosis.FIGS. 45A, 45B, 45C, 45D, 45E, and 45F show the effect of CtBP2overexpression on lipogenic gene expression in wild-type primaryhepatocytes (FIGS. 45A, 45B, 45C, and 45D, n=4) or FoxO1 knockouthepatocytes (FIGS. 45E and 45F, n=5). FIGS. 45G, 45H, 45I, and 45J showresults from liver samples analyzed after 14 days of Ad/shCtBP2transduction in wild-type lean mice. FIG. 45G shows representativeHematoxylin and Eosin stained liver sections. FIG. 45H shows livertriglyceride content (n=6). FIGS. 45I and 45J show liver Stearyol-CoAdesaturase-1 (Scd1) and Fatty acid synthase (Fasn) expression (n=6),respectively. Mice were sacrificed after overnight fasting. Data areexpressed as the mean±SEM. * and ** denote p<0.05 and p<0.01,respectively, determined by Student's t-test.

FIG. 46: Illustrates that the addition of recombinant aP2 activatesFOXO1 translocation and activity. FIG. 46A illustrates nucleartranslocation of FOXO1 in mouse and human islets treated with 10 ug/mlaP2 for 20 minutes (N=3 mouse islets, N=1 human islets). FIG. 46Billustrates gene expression changes of FOXO1 target genes in INS1 cellstreated with 10 ug/ml aP2 for 24 hrs (N=4/group). FIG. 46C illustratesvalidation of change in FOXO1 target gene expression showing increasedVEGFA protein after 24 hr of 10 ug/ml aP2 treatment (N=3/group).

FIG. 47 is a line graph of airway resistance (cmH2O.s/ml) vs.methacholine (mg/ml) during a methacholine challenge test in wild typeand aP2 knockout mice treated with either ovalbumin or vehicle.

DETAILED DESCRIPTION OF THE INVENTION

Anti-aP2 monoclonal antibodies and antigen binding agents are providedthat have superior and unexpected activity for the treatment ofaP2-mediated disorders. For example, anti-aP2 monoclonal antibodies andantigen binding agents are provided that comprise a light chain or lightchain fragment having a variable region, wherein said variable regioncomprises one, two, or three CDRs independently selected from Seq. IDNo. 7, Seq. ID No. 8 and Seq. ID No. 9, Seq. ID No. 10, Seq. ID No. 11,Seq. ID No. 12, and Seq. ID No. 13. Alternatively, one or more of thedisclosed and selected CDRs can be altered by substitution of one ormore amino acids that do not adversely affect or that improve theproperties of the antibody or antigen binding agent, as furtherdescribed herein. In one embodiment, the selected CDR(s) is/are placedin a human immunoglobulin framework. In one embodiment, the humanimmunoglobulin framework is further modified or altered to maintain thebinding affinity specificity of the grafted CDR region.

One of the unexpected discoveries disclosed herein is that the describedantibodies and antigen binding agents do not tightly bind aP2 protein.Typically, antibodies and other antigen binding agents are sought thathave tight binding affinity (very low KD), as was reported by Miao, etal. (See Background of the Invention).

Therefore, in another embodiment, it has been discovered that anantibody or antigen binding agent that binds to aP2 protein in itssecreted (non-cytosolic) state with a weaker binding *

affinity of KD about ≧10⁻⁷ M, has an improved ability to neutralizesecreted aP2 and cause a significant inhibitory effect on aP2-mediateddisorders when provided in an effective amount to a host in needthereof. Furthermore, it has been discovered that the use of alow-affinity binding anti-aP2 antibody reduces the undesirable effectsseen with the use of high affinity anti-aP2 antibodies, for example,weight gain and increased aP2 serum levels.

The anti-aP2 antibodies and antigen binding agents of the presentinvention can alternatively be described by contact points between theantibody or antigen binding agent with the epitope(s) of the aP2protein. aP2 is known to have a discontinuous epitope, in which theamino acids are in close proximity in the folded protein but not closewhen the protein is unfolded or stretched out (see WO 2010/102171).Thus, in one embodiment, the anti-aP2 monoclonal antibody or antigenbinding agent thereof binds an epitope of human aP2 comprising one ormore, for example 1, 2, 3, 4, 5, 6, 7, 8, or 9, amino acid residuesselected from 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, 132A (bolded inSeq. ID No. 1, above), or an amino acid residue within about 3 or 4angstroms of any of 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, and 132A,optionally with a KD for secreted aP2 of about ≧10⁻⁷ M. In a particularembodiment, the antibody has contact with each of these amino acidswithin a 3 or 4 Angstrom range. In another embodiment, the antibody orantigen binding agent of the present invention has 3 or 4 Angstrom rangecontact with at least 6, 7, or 8 of the listed amino acid residues.

In one embodiment, the anti-aP2 monoclonal antibody or antigen bindingagent binds to aP2 only through, or primarily through, light chaincomplementarity determining regions (CDRs). In an alternativeembodiment, the anti-aP2 monoclonal antibody or antigen binding agenthas light chain CDRs that bind to aP2 with a greater affinity than itsheavy chain CDRs bind to aP2. As one example, the antibody or antigenbinding agent specifically binds aP2, and does not specifically bind toFABP5/Mal1.

When administered to a host in need thereof, these anti-aP2 antibodiesand antigen binding agents neutralize the activity of aP2 and providelower fasting blood glucose levels, improved systemic glucosemetabolism, increased systemic insulin sensitivity, reduced fat mass,liver steatosis, improved serum lipid profiles, and/or reducedatherogenic plaque formation in a host when compared to anti-aP2monoclonal antibodies having higher binding affinities. Therefore, theanti-aP2 antibodies and antigen binding agents described herein areparticularly useful to treat metabolic disorders including, but notlimited to, diabetes (both type 1 and type 2), hyperglycemia, obesity,fatty liver disease, dyslipidemia, polycystic ovary syndrome (POS), aproliferative disorder such as a tumor or neoplasm, (including, forexample, transitional bladder cancer, ovarian cancer and liposarcoma),atherosclerosis and other cardiovascular disorders by administering aneffective amount to a host, typically a human, in need thereof.

The present invention thus provides at least the following:

-   -   (a) A monoclonal anti-aP2 antibody or antigenic binding agent,        as described herein, or a described variant or conjugate        thereof.    -   (b) A humanized monoclonal anti-aP2 antibody or antigenic        binding agent, as described herein, or a described variant or        conjugate thereof.    -   (c) A monoclonal anti-aP2 antibody or antigenic binding agent,        as described herein, or a described variant or conjugate        thereof, wherein the antibody or antibody conjugate is        characterized by at least one of:        -   i. Structural inclusion of one or more CDRs described in            Seq. IDs 7-13 or a variant thereof with amino acid            substitutions that do not adversely affect the binding            properties of the CDR region as described in Seq. IDs 7-13;        -   ii. KD binding affinity for human aP2 of ≧10⁻⁷ M;            and/or iii. Contact points with a human or mouse aP2 protein            within 3 or 4 Angstroms as further specified herein.    -   (d) A monoclonal anti-aP2 antibody or antigenic binding agent,        as described herein, or a described variant or conjugate thereof        for use to treat a host, and in particular a human, with an        aP2-mediated disorder.    -   (e) Use of a monoclonal anti-aP2 antibody or antigenic binding        agent, as described herein, or a variant or conjugate thereof,        to treat a host, and in particular a human, with an aP2-mediated        disorder.    -   (f) Use of a monoclonal anti-aP2 antibody or antigenic binding        agent, as described herein, or a variant or conjugate thereof,        in the manufacture of a medicament to treat a host, and in        particular a human, with an aP2-mediated disorder.    -   (g) A process for manufacturing a medicament intended for the        therapeutic use in treating an aP2-mediated disorder,        characterized in that a monoclonal anti-aP2 antibody or        antigenic binding agent, as described herein, or a variant or        conjugate thereof is used in the manufacture.    -   (h) A pharmaceutical composition that includes an effective        amount of a monoclonal anti-aP2 antibody or antigenic binding        agent, as described herein, or a described variant or conjugate        thereof.

GENERAL DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including”, as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit unless specifically stated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics, and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal, andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

That the present invention may be more readily understood, selectedterms are defined below.

The term “host” as used herein, typically refers to a human subject, andin particular where a human or humanized framework is used as anacceptor structure. Where another host is treated, it is understood bythose of skill in the art that the antibody or antigen binding agent mayneed to be tailored to that host to avoid rejection or to make morecompatible. It is known how to use the CDRs in the present invention andengineer them into the proper framework or peptide sequence for desireddelivery and function for a range of hosts. Other hosts may includeother mammals or vertebrate species. The term “host,” therefore, canalternatively refer to animals such as mice, monkeys, dogs, pigs,rabbits, domesticated swine (pigs and hogs), ruminants, equine, poultry,felines, murines, bovines, canines, and the like, where the antibody orantigen binding agent, if necessary is suitably designed forcompatibility with the host.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments, and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The term “human aP2 protein” or “human FABP4/aP2 protein”, as usedherein refers to the protein encoded by Seq. ID. No. 1, and naturalvariants thereof, as described by Baxa, C. A., Sha, R. S., Buelt, M. K.,Smith, A. J., Matarese, V., Chinander, L. L., Boundy, K. L., Bernlohr,A. Human adipocyte lipid-binding protein: purification of the proteinand cloning of its complementary DNA. Biochemistry 28: 8683-8690, 1989.

The term “mouse aP2 protein” or “mouse FAB4P/aP2 protein”, as usedherein, refers to the protein encoded by Seq. ID. No. 2, and naturalvariants thereof. The mouse protein is registered in Swiss-Prot underthe number P04117.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an “antigenicdeterminant” or “epitope” as defined below) on the chemical species, forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally. If an antibody is specificfor epitope “A”, the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains at least someportion of the epitope binding features of an Ig molecule allowing it tospecifically bind to aP2. Such mutant, variant, or derivative antibodyformats are known in the art and described below. Nonlimitingembodiments of which are discussed below. An antibody is said to be“capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.

A “monoclonal antibody” as used herein is intended to refer to apreparation of antibody molecules, which share a common heavy chain andcommon light chain amino acid sequence, or any functional fragment,mutant, variant, or derivation thereof which retains at least the lightchain epitope binding features of an Ig molecule, in contrast with“polyclonal” antibody preparations that contain a mixture of differentantibodies. Monoclonal antibodies can be generated by several knowntechnologies like phage, bacteria, yeast or ribosomal display, as wellas classical methods exemplified by hybridoma-derived antibodies (e.g.,an antibody secreted by a hybridoma prepared by hybridoma technology,such as the standard Kohler and Milstein hybridoma methodology ((1975)Nature 256:495-497).

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region (CH). The heavy chain constant region is comprisedof four domains—either CH1, Hinge, CH2, and CH3 (heavy chains γ, α andδ), or CH1, CH2, CH3, and CH4 (heavy chains μ and ε). Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region (CL). The light chainconstant region is comprised of one domain, CL. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen binding agent” or “antigenic binding agent” as usedherein, refers to one or more fragments or portions of an antibody thatretain the ability to specifically bind to an antigen (e.g., aP2), orsynthetic modifications of antibody fragments that retain the desiredbinding ability to the antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments orcertain portions of a full-length antibody, or modifications thereof.Embodiments include bispecific, dual specific and multi-specific formatswhich may specifically bind to two or more different antigens or toseveral epitopes or discontinuous epitope regions of an antigen.Nonlimiting examples of antigen binding agents include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL, and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al.,PCT publication WO 90/05144 A1 herein incorporated by reference), whichcomprises a single variable domain; (vi) an isolated complementaritydetermining region (CDR), (vii) fusions of antibody fragments such asthose that are immunoglobulin in character, for example, diabodies,scab, bispecific, triabody, Fab-Fv, Fab-Fv-Fv, tribody, (Fab-Fv)2-Fc,and (viii) antibody portions such as CDRs or antibody loops grafted ontonon-immunoglobulin frameworks such as fibronectin or leucine zippers(see Binz et al. (2005) Nat. Biotech. 23:1257-1268, incorporatedherein). Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, usingrecombinant or other methods, by a synthetic or naturally occurringlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also intended to be encompassed within theterm antigen binding agent. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123). Such antibody binding portions are known in theart (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more of the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain, for example ahuman IgA, IgD, IgE, IgG or IgM constant domains. Heavy chain and lightchain constant domain amino acid sequences are known in the art.Non-limiting examples of Ig heavy chain γ1 constant region and Ig lightchain λ and κ chains are provided for in Tables 8 and 6, respectively.

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds aP2 is substantially free of antibodies that specifically bindantigens other than aP2). An isolated antibody that specifically binds,for example, human aP2 may, however, have cross-reactivity to otherantigens, such as aP2 molecules from other species. Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving human heavy and light chain variable regions in which one or moreof the human CDRs (e.g., CDR3) has been replaced with murine CDRsequences.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102(CDR-H3) according to the Kabat numbering system. However, according toChothia (Chothia et al., (1987) J. Mol. Biol., 196, 901-917 (1987)), theloop equivalent to CDR-H1 extends from residue 26 to residue 32. Thusunless indicated otherwise “CDR-H1” as employed herein is intended torefer to residues 26 to 35, as described by a combination of the Kabatnumbering system and Chothia's topological loop definition. For thelight chain variable region, the hypervariable region ranges from aminoacid positions 24 to 34 for CDRL1, amino acid positions 50 to 56 forCDRL2, and amino acid positions 89 to 97 for CDRL3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDRH1, CDRH2 and CDRH3 for the heavy chain CDRs, andCDRL1, CDRL2, and CDRL3 for the light chain CDRs. The term “CDR set” asused herein refers to a group of three CDRs that occur in a singlevariable region capable of binding the antigen. The exact boundaries ofthese CDRs have been defined differently according to different systems.The system described by Kabat (Kabat et al., Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987) and (1991)) not only provides an unambiguous residue numberingsystem applicable to any variable region of an antibody, but alsoprovides precise residue boundaries defining the three CDRs. These CDRsmay be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk,J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883(1989)) found that certain sub-portions within Kabat CDRs adopt nearlyidentical peptide backbone conformations, despite having great diversityat the level of amino acid sequence. These sub-portions were designatedas L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designatesthe light chain and the heavy chains regions, respectively. Theseregions may be referred to as Chothia CDRs, which have boundaries thatoverlap with Kabat CDRs. Other boundaries defining CDRs overlapping withthe Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995))and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDRboundary definitions may not strictly follow one of the above systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat or Chothia, or a mixture thereof,defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987), Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone conformationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human light chain and heavychain acceptor sequences are selected from the sequences described inTables 4, 5, and 7. Different combinations for human framework sequencesFR1 to FR4 are described in said tables.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001).One of the advantages provided by various embodiments of the presentinvention takes advantage of the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

The term “humanized antibody” generally refers to antibodies whichcomprise heavy and light chain variable region sequences from anon-human species (e.g., a rabbit, mouse, etc.) but in which at least aportion of the VH and/or VL sequence has been altered to be more“human-like”, i.e., more similar to human germline variable sequences.One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. Another type ofhumanized antibody is a CDR-grafted antibody, in which at least onenon-human CDR is inserted into a human framework. The latter istypically the focus of the present invention.

In particular, the term “humanized antibody” as used herein, is anantibody or a variant, derivative, analog or fragment thereof whichimmuno-specifically binds to an antigen of interest and which comprisesa framework (FR) region having substantially the amino acid sequence ofa human antibody and a complementarity determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 50, 55, 60, 65, 70, 75 or 80%,preferably at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% identical to the amino acid sequence of a non-human antibodyCDR. In one embodiment, the humanized antibody has a CDR region havingone or more (for example 1, 2, 3 or 4) amino acid substitutions,additions and/or deletions in comparison to the non-human antibody CDR.Further, the non-human CDR can be engineered to be more “human-like” orcompatible with the human body, using known techniques. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′)2, F(ab′)c, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. Preferably, a humanized antibody also comprises at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsboth the light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, and CH3, orCH1, CH2, CH3, and CH4 of the heavy chain. In some embodiments, ahumanized antibody only contains a humanized light chain. In someembodiments, a humanized antibody only contains a humanized heavy chain.In specific embodiments, a humanized antibody only contains a humanizedvariable domain of a light chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgY, IgM, IgG, IgD, IgA and IgE, and anyisotype, including without limitation IgA1, IgA2, IgG1, IgG2, IgG3 andIgG4. The humanized antibody may comprise sequences from more than oneclass or isotype, and particular constant domains may be selected tooptimize desired effector functions using techniques well known in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond exactly toeither the donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95%, 98% or 99% ofthe humanized antibody residues will correspond to those of the parentalFR and CDR sequences. In one embodiment, one or more (for example 1, 2,3 or 4) amino acid substitutions, additions and/or deletions may bepresent in the humanized antibody compared to the parental FR and CDRsequences. As used herein, the term “consensus framework” refers to theframework region in the consensus immunoglobulin sequence. As usedherein, the term “consensus immunoglobulin sequence” refers to thesequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related immunoglobulin sequences (See e.g.,Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany1987). In a family of immunoglobulins, each position in the consensussequence is occupied by the amino acid occurring most frequently at thatposition in the family. If two amino acids occur equally frequently,either can be included in the consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of aP2 protein, for example, secreted aP2 protein,when an antibody described herein specifically binds the aP2 protein.Neutralizing may be the result of different ways of binding of saidantibody to aP2. Preferably a neutralizing antibody is an antibody whosebinding to aP2 results in neutralization of a biological activity ofaP2. Preferably the neutralizing binding protein binds aP2 and decreasesa biologically activity of aP2 by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 60%, 80%, 85%, or more. Neutralization of abiological activity of aP2 by a neutralizing antibody can be assessed bymeasuring one or more indicators of aP2 biological activity describedherein.

A “neutralizing monoclonal antibody” as used herein is intended to referto a preparation of antibody molecules, which upon binding to aP2 areable to inhibit or reduce the biological activity of aP2 eitherpartially or fully.

As used herein, the term “attenuation,” “attenuate,” and the like refersto the lessening or reduction in the severity of a symptom or conditioncaused by elevated serum aP2 levels.

The term “epitope” or “antigenic determinant” includes any polypeptidedeterminant capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three dimensional structural characteristics, and/orspecific charge characteristics. An epitope is a region of an antigenthat is bound by an antibody. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules.

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The strength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller k_(d) represents a greater or higheraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodinvolves measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (“K_(on)”) and the “offrate constant” (“K_(off)”) can be determined by calculation of theconcentrations and the actual rates of association and dissociation.(Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). Davies et al. (1990) Annual Rev Biochem59:439-473.

The term “KD”, as used herein, is intended to refer to the Affinity (orAffinity constant), which is a measure of the rate of binding(association and dissociation) between the antibody and antigen,determining the intrinsic binding strength of the antibody bindingreaction.

The term “antibody conjugate” refers to a binding protein, such as anantibody or antibody fragment or binding portion thereof, chemicallylinked to a second chemical moiety, such as a therapeutic or cytotoxicagent. The term “agent” is used herein to denote a chemical compound, amixture of chemical compounds, a biological macromolecule, or an extractmade from biological materials.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein means a polynucleotide(e.g., of genomic, cDNA, or synthetic origin, or some combinationthereof) that, by virtue of its origin, the “isolated polynucleotide” isnot associated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences, which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation,” as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells, which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.prophylactic or therapeutic agent).

aP2 Protein

Fatty acid-binding proteins (FABPs) are members of the superfamily oflipid-binding proteins (LBP). Nine different FABPs have to date beenidentified, each showing relative tissue enrichment: L (liver), I(intestinal), H (muscle and heart), A (adipocyte), E (epidermal), Il(ileal), B (brain), M (myelin) and T (testis). The primary role of allthe FABP family members is regulation of fatty acid uptake andintracellular transport. The structure of all FABPs is similar—the basicmotif characterizing these proteins is β-barrel, and a single ligand(e.g. a fatty acid, cholesterol, or retinoid) is bound in its internalwater-filled cavity.

The adipocyte fatty acid-binding protein aP2 regulates systemic glucoseand lipid metabolism, and has been implicated in the pathology of manyimmunometabolic diseases, such as diabetes and atherosclerosis. WhileaP2 has classically been considered a cytosolic protein, it has beenfound to be an active adipokine that contributes to hyperglycemia bypromoting hepatic gluconeogenesis. Serum aP2 levels have been found tobe markedly elevated in mouse and human obesity.

The human aP2 protein is a 14.7 kDa intracellular and extracellular(secreted) lipid binding protein that consists of 132 amino acidscomprising the amino acid sequence (Seq. ID No. 1) of Table 1. The cDNAsequence of human aP2 was previously described in Baxa, C. A., Sha, R.S., Buelt, M. K., Smith, A. J., Matarese, V., Chinander, L. L., Boundy,K. L., Bernlohr, A. Human adipocyte lipid-binding protein: purificationof the protein and cloning of its complementary DNA. Biochemistry 28:8683-8690, 1989, and is provided in Seq. ID No. 5. The human protein isregistered in Swiss-Prot under the number P15090.

The mouse aP2 protein sequence comprises the amino acid sequence of Seq.ID No. 2 of Table 1. The cDNA sequence of mouse aP2 is provided in Seq.ID No. 6. The mouse protein is registered in Swiss-Prot under the numberP04117.

Both the human and mouse aP2 protein include at least two majorconserved domains: an 11 amino acid nuclear localization signal(aa22-32: kevgvgfatrk (Seq. ID No. 3)); and a three amino acid fattyacid binding region (aa127-129: rvy (Seq. ID No. 4)).

TABLE 1 aP2 Protein and cDNA Sequences Protein or cDNA Seq. ID No.SEQUENCE Fatty acid-binding protein, 1 MCDAFVGTWKLVSSENFDDYMKEVGVGFATRKVadipocyte (FABP4/aP2)[H. sapiens] AGMAKPNMIISVNGDVITIKSESTFKNTEISFILGQEFDEVTADDRKVKSTITLDGGVLVHVQKWDGKSTT IKRKREDDKLVVECVMKGVTSTRVYERA Fattyacid-binding protein, 2 MCDAFVGTWKLVSSENFDDYMKEVGVGFATRKV adipocyte(FABP4/aP2 [M. musculus]) AGMAKPNMIISVNGDL VTIRSESTFKNTEISFKLGVEFDEITADDRKVKSIITLDGGALVQVQKWDGKSTTI KRKRDGDKLVVECVMKGVTSTRVYERA aP2nuclear localization amino 3 KEVGVGFATRK acid sequence aP2 fatty acidbinding domain 4 RVY amino acid sequence Fatty acid-binding protein, 5ATGTGTGATGCTTTTGTAGGTACCTGGAAACTTG adipocyte (FABP4/aP2)[H. sapiens]TCTCCAGTGAAAACTTTGATGATTATATGAAAGA cDNAAGTAGGAGTGGGCTTTGCCACCAGGAAAGTGGC TGGCATGGCCAAACCTAACATGATCATCAGTGTGAATGGGGATGTGATCACCATTAAATCTGAAAGT ACCTTTAAAAATACTGAGATTTCCTTCATACTGGGCCAGGAATTTGACGAAGTCACTGCAGATGACA GGAAAGTCAAGAGCACCATAACCTTAGATGGGGGTGTCCTGGTACATGTGCAGAAATGGGATGG AAAATCAACCACCATAAAGAGAAAACGAGAGGATGATAAACTGGTGGTGGAATGCGTCATGAAAG GCGTCACTTCCACGAGAGTTTATGAGAGAGCAT AAFatty acid-binding protein, 6 ATGTGTGATGCCTTTGTGGGAACCTGGAAGCTTGadipocyte (FABP4/aP2 [M. musculus]) TCTCCAGTGAAAACTTCGATGATTACATGAAAGAcDNA AGTGGGAGTGGGCTTTGCCACAAGGAAAGTGGC AGGCATGGCCAAGCCCAACATGATCATCAGCGTAAATGGGGATTTGGTCACCATCCGGTCAGAGAG TACTTTTAAAAACACCGAGATTTCCTTCAAACTGGGCGTGGAATTCGATGAAATCACCGCAGACGAC AGGAAGGTGAAGAGCATCATAACCCTAGATGGCGGGGCCCTGGTGCAGGTGCAGAAGTGGGATGGA AAGTCGACCACAATAAAGAGAAAACGAGATGGTGACAAGCTGGTGGTGGAATGTGTTATGAAAGGC GTGACTTCCACAAGAGTTTATGAAAGGGCATGAaP2-Binding Epitopes

In one aspect of the invention, anti-aP2 monoclonal antibody molecules,including humanized monoclonal antibodies, and antigen binding agentsare provided, that specifically bind to human aP2 or mouse aP2 atspecific, identified amino acids within the aP2 molecule while folded inits native, conformational form or complexed with its natural bindingpartner.

In one embodiment, the anti-aP2 monoclonal antibody binds human aP2having the amino acid sequence:

(Seq. ID. No 1) MCDAFVGTWK LVSSENFDDY MKEVGVGFAT RKVAGMAKPN MIISVNGDVITIKSESTFKN TEISFILGQE FDEVTADDRK VKSTITLDGG VLVHVQKWDG KSTTIKRKREDDKLVVECVM KGVTSTRVYE RA ,or a naturally occurring variant thereof.

In an alternative embodiment, the anti-aP2 monoclonal antibody orantigen binding agent binds to a human aP2 having an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSeq. ID No. 1. In one embodiment, the antibody has a KD for aP2 of ≧10⁻⁷M. In one embodiment, the antibody binds to an epitope selected from anamino acid sequence underlined in Seq. ID No. 1 above and has a KD foraP2 of about ≧10⁻⁷ M. In one embodiment, the antibody binds to anepitope that has one or more (for example, 1, 2, 3, or 4) amino acidsubstitutions, additions, or deletions as compared to Seq. ID. No. 1.

aP2-Binding Epitopes as Determined by X-Ray Crystallography

In one embodiment, the anti-aP2 antibody or antigen binding agentdirectly interacts with one or more, for example 1, 2, 3, 4, 5, 6, 7, 8,or 9, amino acids bolded in Seq. ID No. 1 above within a 3 or 4 Angstromdistance. In another embodiment, the anti-aP2 monoclonal antibody orantigen binding agent contacts all nine of the bolded amino acids inSeq. ID No. 1 above within a 3 or 4 Angstrom distance.

In one embodiment, the purified anti-aP2 monoclonal antibody binds anon-contiguous epitope of human and/or mouse aP2 comprising at least oneor more, for example 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residuesselected from 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, and 132A (boldedin Seq. ID No. 1, above), or an amino acid residue within about 4angstroms of any of 10K, 11L, 12V, 13S, 37A, 38K, 57T, 130E, and 132A.In one embodiment, the purified anti-aP2 monoclonal antibody binds anepitope of human aP2 comprising at least one or more, for example 1, 2,3, 4, 5, 6, 7, 8, or 9, amino acid residues selected from 10K, 11L, 12V,13S, 37A, 38K, 57T, 130E, and 132A (bolded in Seq. ID No. 1, above), oran amino acid residue within about 4 angstroms of any of 10K, 11L, 12V,13S, 37A, 38K, 57T, 130E, and 132A and has a KD for aP2 of ≧10⁻⁷ M.

In an alternative embodiment, the purified anti-aP2 monoclonal antibodybinds an epitope of human and/or mouse aP2 comprising at least one ormore, for example 1, 2, 3, 4, 5, 6, or 7, amino acid residues selectedfrom 10K, 11L, 12V, 13S, 38K, 130E, or 132A (bolded in Seq. ID No. 1,above), or an amino acid residue within about 4 angstroms of any of 10K,11L, 12V, 13S, 38K, 130E, or 132A. In one embodiment, the purifiedanti-aP2 monoclonal antibody binds an epitope of human aP2 comprising atleast one or more, for example 1, 2, 3, 4, 5, 6, or 7, amino acidresidues selected from 10K, 11L, 12V, 13S, 38K, 130E, or 132A (bolded inSeq. ID No. 1, above), or an amino acid residue within about 4 angstromsof any of 10K, 11L, 12V, 13S, 38K, 130E, or 132A and has a KD for aP2 of≧10⁻⁷ M. In one embodiment, the antibody further binds 37A and/or 57T.

In one embodiment, the light chain of the antibody binds an epitope ofhuman aP2 comprising at least one or more, for example 1, 2, 3, 4, 5, 6,7, 8, or 9, amino acid residues selected from 10K, 11L, 12V, 13S, 37A,38K, 57T, 130E, or 132A, or an amino acid residue within about 4angstroms thereof, and has a KD of at least about ≧10⁻⁷ M. In oneembodiment, the light chain of the antibody binds an epitope of humanaP2 comprising at least one or more, for example 1, 2, 3, 4, 5, 6, or 7,amino acid residues selected from 10K, 11L, 12V, 13S, 37A, 38K, 57T,130E, or 132A, or an amino acid residue within about 4 angstromsthereof, and has a KD of at least about ≧10⁷⁻⁷ M.

In one embodiment, the light chain of the antibody binds an epitope ofhuman aP2 comprising at least one or more, for example 1, 2, 3, 4, 5, 6,7, 8, or 9, amino acid residues selected from 10K, 11L, 12V, 13S, 37A,38K, 57T, 130E, or 132A, or an amino acid residue within about 4angstroms thereof, and the heavy chain of the antibody binds an epitopeof human aP2 comprising at least one of 10K and 132A, or an amino acidwithin about 4 angstroms thereof. In one embodiment, the antibody has aKD of at least about ≧10⁻⁷ M.

In one embodiment, the light chain of the antibody binds an epitope ofhuman aP2 comprising at least one or more, for example 1, 2, 3, 4, 5, 6,or 7, amino acid residues selected from 10K, 11L, 12V, 13S, 38K, 130E,or 132A, or an amino acid residue within about 4 angstroms thereof, andthe heavy chain of the antibody binds an epitope of human aP2 comprisingat least one of 10K and 132A, or an amino acid within about 4 angstromsthereof, and has a KD of at least about ≧10⁻⁷ M.

In one embodiment, the antibody binds to the human aP2 protein in itsnative confirmation at least at 10K. In one embodiment, the antibodybinds to the human aP2 protein in its native confirmation at least at38K. In one embodiment, the antibody binds to the human aP2 protein inits native confirmation at least at 12V. In one embodiment, the antibodybinds to the human aP2 protein in its native confirmation at least at11L. In one embodiment, the antibody binds to the human aP2 protein inits native confirmation at least at 130E. In one embodiment, theantibody binds to the human aP2 protein in its native confirmation atleast at 132A. In one embodiment, the antibody binds to the human aP2protein in its native confirmation at least at 13S. In one embodiment,the antibody binds to the human aP2 protein in its native confirmationat least at 11L and 12V. In one embodiment, the antibody binds to thehuman aP2 protein in its native confirmation at least at 130E and 132A.In one embodiment, the antibody binds to the human aP2 protein in itsnative confirmation at a specific amino acid described above and has aKD of about ≧10⁻⁷ M.

In one embodiment, the antibody binds to the human aP2 protein in itsnative confirmation at least at 10K and 38K. In one embodiment, theantibody binds to the human aP2 protein in its native confirmation atleast at 10K, 38K, and 12V. In one embodiment, the antibody binds to thehuman aP2 protein in its native confirmation at least at 10K, 38K, 12V,and 11L. In one embodiment, the antibody binds to the human aP2 proteinin its native confirmation at least at 10K, 38K, 12V, 11L, and 57T. Inone embodiment, the antibody binds to the human aP2 protein in itsnative confirmation at least at 10K, 38K, 12V, 11L, 57T, and 37A. In oneembodiment, the antibody binds to the human aP2 protein in its nativeconfirmation at least at 10K, 38K, 12V, 11L, 57T, 37A, and 130E. In oneembodiment, the antibody binds to the human aP2 protein in its nativeconfirmation at least at 10K, 38K, 12V, 11L, 57T, 37A, 130E, and 132A.In one embodiment, the antibody binds to the human aP2 protein in itsnative confirmation at least at 10K, 38K, 12V, 11L, 57T, 37A, 130E,132A, and 13S.

Also provided herein is a specific region or epitope of human aP2 whichis bound by an antibody provided by the present invention, in particularan epitope bound by the antibody comprising the light chain variablesequence 909gL1 (Seq. ID No. 446), 909gL10 (Seq. ID No. 448), 909gL13(Seq. ID No. 487), 909gL50 (Seq. ID No. 488), 909gL54 (Seq. ID No. 450),or 909gL55 (Seq. ID No. 452), and/or heavy chain variable sequence909gH1 (Seq. ID No. 455), 909gH14 (Seq. ID No. 457), 909gH15 (Seq. IDNo. 459), 909gH61 (Seq. ID No. 461), or 909gH62 (Seq. ID No. 463).

This specific region or epitope of the human aP2 protein provided hereincan be identified by any suitable epitope mapping method known in theart in combination with any one of the antibodies provided by thepresent invention. Examples of such methods include screening peptidesof varying lengths derived from aP2 for binding to the antibody of thepresent invention with the smallest fragment that can specifically bindto the antibody containing the sequence of the epitope recognized by theantibody. The aP2 peptides may be produced synthetically or byproteolytic digestion of the aP2 protein. Peptides that bind theantibody can be identified by, for example, mass spectrometric analysis.In another example, NMR spectroscopy or X-ray crystallography can beused to identify the epitope bound by an antibody of the presentinvention. Crystallization and X-ray crystallography techniques fordetermining the structure of aP2 and specific interactions of the aP2protein with its natural binding partners, for example medium chain andlong chain fatty acids, are described in Marr et al., Expression,purification, crystallization and structure of human adipocytelipid-binding protein (aP2), Acta Cryst. (2006), F62, 1058-1060. Onceidentified, the epitopic fragment which binds an antibody of the presentinvention can be used, if desired, as an immunogen to obtain additionalantibodies which bind the same epitope.

In one example the epitope of the antibody molecule is determined byX-ray crystallography using the aP2 protein (Seq. ID No. 1).

In one embodiment, the antibody of the present invention comprises atleast one or more specific amino acids within a CDR domain as defined inTable 2 that interact with a mouse or human aP2 protein in its nativeconformation at the amino acid contact point defined in Table 2.

TABLE 2 Anti-aP2 antibody/aP2 protein Contact Points Ab Amino Ab SourceaP2 Amino aP2 Target Distance Ab Chain Ab CDR Acid Atom Acid Atom(angstroms) Light CDRL3 92Tyr C 10Lys C 3.72 Light CDRL3 92Tyr C 10Lys C3.92 Light CDRL3 92Tyr O 10Lys C 3.66 Light CDRL3 93Gly N 10Lys C 3.30Light CDRL3 93Gly C 10Lys C 3.65 Light CDRL3 93Gly C 10Lys C 3.67 LightCDRL3 94Thr N 10Lys C 3.74 Light CDRL3 92Tyr C 10Lys C 3.25 Light CDRL392Tyr C 10Lys C 3.85 Light CDRL3 92Tyr C 10Lys C 3.14 Light CDRL3 94ThrO 10Lys C 3.31 Heavy 104Leu* C 10Lys N 3.06 Light CDRL3 93Gly N 10Lys N2.74 Light CDRL3 93Gly C 10Lys N 3.64 Light CDRL3 92Tyr C 10Lys N 3.37Light CDRL3 92Tyr C 10Lys N 3.32 Light CDRL3 92Tyr C 10Lys N 3.02 LightCDRL3 92Tyr C 10Lys N 3.87 Light CDRL3 92Tyr C 10Lys N 3.35 Light CDRL392Tyr C 10Lys N 3.88 Light CDRL3 92Tyr C 10Lys N 3.29 Light CDRL3 92TyrC 10Lys N 3.82 Light CDRL3 92Tyr O 11Leu N 3.77 Light CDRL3 11Leu C 3.93Light CDRL3 11Leu C 3.95 Light CDRL3 11Leu C 3.46 Light CDRL3 92Tyr C11Leu O 3.51 Light CDRL3 92Tyr C 11Leu O 3.82 Light CDRL3 92Tyr O 11LeuO 2.40 Light CDRL3 95Tyr C 11Leu O 3.60 Light CDRL3 12Val C 3.88 LightCDRL3 12Val C 3.52 Light CDRL3 96Ala N 12Val C 3.92 Light CDRL3 95Tyr C12Val C 3.88 Light CDRL3 95Tyr C 12Val O 3.25 Light CDRL3 95Tyr C 12ValO 3.43 Light CDRL3 95Tyr C 12Val O 3.45 Light CDRL3 96Ala N 12Val O 2.72Light CDRL3 96Ala C 12Val O 3.77 Light CDRL3 95Tyr C 12Val O 3.90 LightCDRL3 95Tyr C 12Val O 3.97 Light CDRL3 13Ser N 3.93 Light CDRL1 28Asp C37Ala C 3.94 Light CDRL1 28Asp C 37Ala C 3.68 Light CDRL1 28Asp O 37AlaC 3.83 Light CDRL1 28Asp O 37Ala C 4.00 Light CDRL1 28Asp C 38Lys N 3.88Light CDRL1 28Asp C 38Lys N 3.60 Light CDRL1 28Asp O 38Lys N 3.20 LightCDRL1 38Lys C 3.93 Light CDRL1 28Asp O 38Lys C 3.40 Light CDRL1 38Lys C3.40 Light CDRL1 27Glu O 38Lys C 3.50 Light CDRL3 95Tyr C 38Lys C 3.80Light CDRL3 95Tyr C 38Lys C 3.83 Light CDRL3 95Tyr C 38Lys N 3.99 LightCDRL1 28Asp O 38Lys N 3.56 Light CDRL1 27Glu C 38Lys N 2.96 Light CDRL395Tyr C 38Lys N 3.54 Light CDRL3 95Tyr C 38Lys N 3.28 Light CDRL3 95TyrC 38Lys N 3.54 Light 100Phe* C 38Lys N 3.23 Light 100Phe* C 38Lys N 3.95Light CDRL1 28Asp O 38Lys O 3.89 Light CDRL1 30Ser O 38Lys O 3.37 LightCDRL1 28Asp O 57Thr C 3.57 Light CDRL1 28Asp C 57Thr O 3.25 Light CDRL128Asp O 57Thr O 2.61 Light CDRL1 28Asp O 57Thr O 3.15 Light CDRL1 28AspO 57Thr C 3.95 Light CDRL3 94Thr O 130Glu C 3.88 Light CDRL3 94Thr C130Glu O 3.84 Light CDRL3 94Thr O 130Glu O 2.81 Heavy CDRH3 104Leu* C132Ala C 3.95 Heavy CDRH3 132Ala C 3.97 Light CDRL1 32Tyr O 132Ala C3.59 *indicates contact points outside of CDR regions as determined byKabat numbering

The anti-aP2 monoclonal antibodies of the present invention can furtherbe defined by specific amino acids within the CDRs that contact the aP2protein in its native, conformational form during binding. In oneembodiment, provided is a purified anti-aP2 monoclonal antibodycomprising a light chain comprising the following amino acids at theidentified specific position: CDRL1—27Glu, 28Asp, 30Ser; CDRL3—92Tyr,93Gly, 94Thr, 95Tyr, 96Ala; and 100Phe. In one embodiment, the antibodybinds to the human aP2 protein in its native confirmation and has a KDof about ≧10⁻⁷ M. In one embodiment, the anti-aP2 monoclonal antibody ishumanized.

In one embodiment, provided is a purified anti-aP2 monoclonal antibodycomprising a light chain comprising the following amino acids at theidentified position: CDRL1—27Glu, 28Asp, 30Ser; CDRL3—92Tyr, 93Gly,94Thr, 95Tyr, 96Ala; and 100Phe; and a heavy chain comprising thefollowing amino acid at the identified position: 104Leu. In oneembodiment, the antibody binds to the human aP2 protein in its nativeconfirmation and has a KD of about ≧10⁻⁷ M. In one embodiment, theanti-aP2 monoclonal antibody is humanized.

In one embodiment, provided is a purified anti-aP2 monoclonal antibodycomprising a light chain comprising the following amino acid at theidentified position: CDRL3—91Ala; and a heavy chain comprising thefollowing amino acids at the identified positions: CDRH1—33Ala;CDRH2—52Ser. In one embodiment, the heavy chain further comprises thefollowing amino acid at the identified position: CDRH3—98Phe. In oneembodiment, the antibody binds to the human aP2 protein in its nativeconfirmation and has a KD of about ≧10⁻⁷ M. In one embodiment, theanti-aP2 monoclonal antibody is humanized.

In one embodiment, the purified anti-aP2 monoclonal antibody binds toaP2 only through, or primarily through, its light chain CDRs. In analternative embodiment, the purified anti-aP2 monoclonal antibody haslight chain CDRs that bind to aP2 with a greater affinity than its heavychain CDRs bind to aP2.

In one embodiment, the purified anti-aP2 monoclonal antibody ischaracterized by having a low affinity for human aP2 in its native,conformational form. In one embodiment, the purified anti-aP2 monoclonalantibody has a KD for human aP2 of about ≧10⁻⁷ M. In one embodiment, thepurified anti-aP2 monoclonal antibody has a KD for human aP2 of betweenabout 10⁻⁴ to 10⁻⁶ M. In one embodiment, the purified anti-aP2monoclonal antibody has a KD for human aP2 of about >500 nM. In oneembodiment, the purified anti-aP2 monoclonal antibody has a KD for humanaP2 of about 500 nM to about 10 μM. In one embodiment, the purifiedanti-aP2 monoclonal antibody has a KD for human aP2 of about 1 μM toabout 7 μM. In one embodiment, the purified anti-aP2 monoclonal antibodyhas a KD for human aP2 of about 2 μM to about 5 μM.

In an alternative embodiment, the purified anti-aP2 monoclonal antibodyhas a low binding affinity for mouse aP2 in its native, conformationalform. In one embodiment, the purified anti-aP2 monoclonal antibody has aKD for mouse aP2 of ≧10⁻⁷ M. In one embodiment, the purified anti-aP2monoclonal antibody has a KD for mouse aP2 of between about 10⁻⁴ to 10⁻⁶M. In one embodiment, the purified anti-aP2 monoclonal antibody has a KDfor mouse aP2 of about >500 nM. In one embodiment, the purified anti-aP2monoclonal antibody has a KD for mouse aP2 of about 500 nM to about 10μM. In one embodiment, the purified anti-aP2 monoclonal antibody has aKD for mouse aP2 of about 1 μM to about 7 μM. In one embodiment, thepurified anti-aP2 monoclonal antibody has a KD for mouse aP2 of about 2μM to about 5 μM.

In one embodiment, the antibody specifically binds aP2, and does notspecifically bind to FABP5/Mal1.

In one embodiment, provided is a purified anti-aP2 monoclonal antibody,and a process of making same, wherein the affinity for aP2 has beenreduced by identifying an anti-aP2 antibody having a KD for aP2 of atleast <10-7 M, altering at least one amino acid in either a CDR regionor a FR region of the anti-aP2 antibody, wherein the alteration resultsin an anti-aP2 antibody with a KD of at least about ≧10⁻⁷ M. In oneembodiment, the altered antibody is capable of binding to human aP2 inits native conformational form. In an alternative embodiment, thealtered antibody is capable of binding to mouse aP2 in its nativeconformation form. In one embodiment, provided is a method of reducingthe affinity for an anti-aP2 antibody having a KD of <10⁻⁷ Mcomprising 1) identifying a monoclonal antibody having an affinity of atleast <10⁻⁷ M for an aP2 protein, 2) identifying an amino acid within aCDR region that contacts the aP2 protein in its native conformationalform, and 3) substituting one or more contact amino acids with an aminoacid selected from alanine, phenylalanine, and serine, wherein thesubstitutions result in a reduction in the affinity of the antibody to aKD of about ≧10⁻⁷ M. In one embodiment, a cysteine residue in CDRL3 issubstituted. In one embodiment, the cysteine residue is substituted withan amino acid selected from alanine, glutamine, and histidine.

aP2-Binding Epitopes as Determined by Hydrogen-Deuterium Exchange

In one aspect of the invention, the purified monoclonal antibody bindsto an epitope of the human aP2 protein as determined byhydrogen-deuterium exchange (HDX) comprising at least one, for example1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, of amino acids 9-17, aminoacids 20-28, or amino acids 118-132 of Seq. ID No. 1. In one embodiment,the purified monoclonal antibody binds to an epitope of the human aP2protein comprising at least one, for example 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more, of amino acids 9-17 (WKLVSSENF) (Seq. ID No. 22), aminoacids 20-28 (YMKEVGVGF) (Seq. ID No. 23), or amino acids 118-132(CVMKGVTSTRVYERA) (Seq. ID No. 24) of Seq. ID No. 1, and has a KD of atleast about ≧10⁻⁷ M. In one embodiment, the purified anti-aP2 monoclonalantibody binds to an epitope of the human aP2 protein within 3-4angstrom contact points of at least one, for example 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more, of amino acids 9-17, amino acids 20-28, or aminoacids 118-132 of Seq. ID No. 1 when the human aP2 protein is in itsnative, conformational form. In one embodiment, the purified anti-aP2monoclonal antibody binds to an epitope of the human aP2 protein within3-4 angstrom contact points of at least one of, for example 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more, amino acids 9-17, amino acids 20-28, or aminoacids 118-132 of Seq. ID No. 1 when the human aP2 protein is in itsnative, conformational form and has a KD of at least about ≧10⁻⁷ M.Hydrogen-deuterium exchange (HDX) to determine binding interactions andantibody-epitope maps are well known in the art, for example, asdescribed by Pandit et al. (2012) J. Mol. Recognit. March; 25(3): 114-24(incorporated herein by reference)).

Anti-aP2 Antibodies and Antigen Binding Agent Structures

Anti-aP2 monoclonal antibodies and antigen binding agents have beendiscovered that have superior and unexpected activity for the treatmentof aP2-mediated disorders. In one embodiment, anti-aP2 monoclonalantibodies and fragments are provided that contain a light chain orlight chain fragment having a variable region, wherein said variableregion comprises one, two or three CDRs independently selected from Seq.ID No. 7, Seq. ID No. 8, and Seq. ID No. 9, Seq. ID No. 10, Seq. ID No.11, Seq. ID No. 12 and Seq. ID No. 13. Alternatively, one or more of thedisclosed and selected CDRs can be altered by substitution of one ormore amino acids (for example, 1, 2, 3, 4, 5, 6, 7 or 8 amino acids)that do not adversely affect or that improve the properties of theantibody or antigen binding agent, as further described herein. In oneembodiment, the selected CDR(s) is/are placed in a human immunoglobulinframework. In one embodiment, the human immunoglobulin framework isfurther modified or altered to maintain the binding affinity specificityof the grafted CDR region.

Therefore, in another embodiment, it has been discovered that anantibody or antigen binding agent that binds to aP2 protein in itssecreted (non-cytosolic) state with a weaker binding affinity of KDabout ≧10⁻⁷ M, has an improved ability to neutralize secreted aP2 andcause a significant inhibitory effect on aP2-mediated disorders whenprovided to in an effective amount to a host in need thereof.

In an antibody molecule, there are two heavy chains and two lightchains. Each heavy chain and each light chain has at its N-terminal enda variable domain. Each variable domain is composed of four frameworkregions (FRs) alternating with three complementarity determining regions(CDRs). The residues in the variable domains are conventionally numberedaccording to a system devised by Kabat et al (supra), or a combinationof Kabat and Chothia as described above for CDR-H1. This numberingsystem is used in the present specification except where otherwiseindicated.

Immunoglobulins (Ig) are the antigen recognition molecules of B cells.An Ig molecule is made up of 2 identical heavy chains and 2 identicallight chains, either kappa or lambda, joined by disulfide bonds so thateach heavy chain is linked to a light chain and the 2 heavy chains arelinked together. The kappa and lambda light chains have no apparentfunctional differences. Each Ig kappa and lambda light chain has anN-terminal variable (V) region containing the antigen-binding site and aC-terminal constant (C) region, encoded by the C region gene (IGKC orIGLC), that provides signaling functions. The kappa and lambda lightchain V regions are encoded by 2 types of genes: V genes and joining (J)genes. Random selection of just 1 gene of each type to assemble a Vregion accounts for the great diversity of V regions among Ig molecules.The kappa light chain locus on human chromosome 2 contains approximately40 functional V genes, followed by approximately 5 functional J genes.The lambda light chain locus on human chromosome 22 containsapproximately 30 functional V genes, followed by approximately 4functional J genes. Due to polymorphism, the numbers of functional V andJ genes differ among individuals.

Each Ig heavy chain has an N-terminal variable (V) region containing theantigen-binding site and a C-terminal constant (C) region, encoded by aC region gene, that provides effector or signaling functions. The heavychain V region is encoded by 3 types of genes: V genes, joining (J)genes, and diversity (D) genes. Random selection of just 1 gene of eachtype to assemble a V region accounts for the great diversity of Vregions among Ig molecules. The heavy chain locus on human chromosome 14contains approximately 40 functional V genes, followed by approximately25 functional D genes and approximately 6 functional J genes. Due topolymorphism, the numbers of functional V, J, and D genes differ amongindividuals. There are five types of mammalian immunoglobulin heavychains: γ, δ, α, μ and ε. They define classes of immunoglobulins: IgG,IgD, IgA, IgM and IgE, respectively. Heavy chains γ, α and δ have aconstant region composed of three tandem (in a line next to each other)immunoglobulin domains but also have a hinge region between CH1 and CH2regions for added flexibility. Heavy chains μ and ε have a constantregion composed of four domains.

The Kabat residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or CDR, of the basicvariable domain structure. The correct Kabat numbering of residues maybe determined for a given antibody by alignment of residues of homologyin the sequence of the antibody with a “standard” Kabat numberedsequence.

The CDRs of the heavy chain variable domain are located at residues31-35 (CDRH1), residues 50-65 (CDRH2) and residues 95-102 (CDRH3)according to the Kabat numbering. However, according to Chothia (Chothiaet al., (1987) J. Mol. Biol., 196, 901-917), the loop equivalent toCDR-H1 extends from residue 26 to residue 32. Thus unless indicatedotherwise, “CDR-H1” as employed herein is intended to refer to residues26 to 35, as described by a combination of the Kabat numbering systemand Chothia's topological loop definition.

The CDRs of the light chain variable domain are located at residues24-34 (CDRL1), residues 50-56 (CDRL2) and residues 89-97 (CDRL3)according to the Kabat numbering. Antibodies for use in the presentdisclosure may be obtained using any suitable method known in the art.The aP2 protein including fusion proteins, or cells (recombinantly ornaturally) expressing the protein, can be used to produce antibodies,which specifically recognize aP2. The aP2 protein used can be the fullbiologically active protein or a fragment or derivative thereof.

aP2 proteins or peptides, for use to immunize a host, may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems or they may be recovered from naturalbiological sources. The aP2 protein may in some instances be part of alarger protein such as a fusion protein for example fused to an affinitytag or similar, or complexed with its naturally occurring biologicalpartner.

Antibodies generated against the aP2 protein may be obtained, whereimmunization of an animal is necessary, by administering the protein toan animal, preferably a non-human animal, using well-known and routineprotocols, see for example Handbook of Experimental Immunology, D. M.Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England,1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep,cows, camels or pigs may be immunized. However, mice, rabbits, pigs andrats are generally most suitable. Monoclonal antibodies may be preparedby any method known in the art such as the hybridoma technique (Kohler &Milstein, 1975, Nature, 256:495-497), the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72)and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies andCancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by, for example, the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA93(15):7843-78481; WO92/02551; WO2004/051268 and International PatentApplication number WO2004/106377. Screening for antibodies can beperformed using assays to measure binding to human aP2 and/or assays tomeasure the ability to block aP2 binding to its natural receptor. Anexample of a binding assay is an ELISA, in particular, using a fusionprotein of human aP2 and human Fc, which is immobilized on plates, andemploying a secondary antibody to detect anti-aP2 antibody bound to thefusion protein. Examples of suitable antagonistic and blocking assaysare described in the Examples herein.

Humanized antibodies (which include CDR-grafted antibodies) are antibodymolecules having one or more complementarity determining regions (CDRs)from a non-human species (e.g., a rabbit or mouse) and a frameworkregion from a human immunoglobulin molecule (see, e.g. U.S. Pat. No.5,585,089; WO91/09967). It will be appreciated that it may only benecessary to transfer the specificity determining residues of the CDRsrather than the entire CDR (see for example, Kashmiri et al., 2005,Methods, 36, 25-34). Humanized antibodies may optionally furthercomprise one or more framework residues derived from the non-humanspecies from which the CDRs were derived. The latter are often referredto as donor residues. The antibody molecules of the present inventionsuitably have a binding affinity of about ≧10⁻⁷ M, in particular in themicromolar (μM) range. Affinity may be measured using any suitablemethod known in the art, including BIAcore, as described in the Examplesherein, using isolated natural or recombinant aP2 or a suitable fusionprotein/polypeptide. In one embodiment described herein, the bindingaffinities of the anti-aP2 monoclonal antibody described herein mayinclude antibodies having a KD of about ≧10⁻⁷ M. In one embodiment, thepurified anti-aP2 monoclonal antibody has a KD for human aP2 of betweenabout 10⁻⁴ to 10⁻⁶ M. In one embodiment, the purified anti-aP2monoclonal antibody has a KD for human aP2 of about 2 to about 5 μM.

In one example the antibody of the present invention does not bindFABP5/Mal1. In one example the antibody of the present invention bindsaP2 in its natural non-linear structural conformation.

The affinity of an antibody or antigen binding agent of the presentinvention, as well as the extent to which a binding agent (such as anantibody) inhibits binding, can be determined by one of ordinary skillin the art using conventional techniques, for example those described byScatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)) or by surfaceplasmon resonance (SPR) using systems such as BIAcore. For surfaceplasmon resonance, target molecules are immobilized on a solid phase andexposed to ligands in a mobile phase running along a flow cell. Ifligand binding to the immobilized target occurs, the local refractiveindex changes, leading to a change in SPR angle, which can be monitoredin real time by detecting changes in the intensity of the reflectedlight. The rates of change of the SPR signal can be analysed to yieldapparent rate constants for the association and dissociation phases ofthe binding reaction. The ratio of these values gives the apparentequilibrium constant (affinity) (see, e.g., Wolff et al, Cancer Res.53:2560-65 (1993)).

In the present invention affinity of the test antibody molecule istypically determined using SPR as follows. The test antibody molecule iscaptured on the solid phase and human aP2 is run over the capturedantibody in the mobile phase and affinity of the test antibody moleculefor human aP2 is determined. The test antibody molecule may be capturedon the solid phase chip surface using any appropriate method, forexample using an anti-Fc or anti Fab′ specific capture agent. In oneexample the affinity is determined at pH 6. In one example the affinityis determined at pH 7.4.

It will be appreciated that the affinity of antibodies provided by thepresent invention may be altered using any suitable method known in theart. The present invention therefore also relates to variants of theantibody molecules of the present invention, which have an improvedaffinity for aP2. Also contemplated, as described further herein, is themodification of high affinity anti-human aP2 antibodies in order toreduce the KD to at least about ≧10⁻⁷ M. Such variants can be obtainedby a number of affinity maturation protocols including mutating the CDRs(Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Markset al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E.coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling(Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phagedisplay (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexualPCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra)discusses these methods of affinity maturation.

CDR Regions

In one aspect of the present invention, an anti-aP2 monoclonal antibodyor antigen binding agent is provided that binds to aP2 protein in itsnative conformation wherein the antibody comprises at least one, or morethan one, of the CDR regions provided in Table 3.

TABLE 3 Anti-aP2 Antibody Complementarity Determining Regions (CDRs)Protein Seq. ID No. SEQUENCE CDRL1 7 QASEDISRYLV CDRL1 variant 1 597SVSSSISSSNLH CDRL2 8 KASTLAS CDRL2 variant 1 598 GTSNLAS CDRL3 9QCTYGTYAGSFFYS CDRL3 variant 1 10 QATYGTYAGSFFYS CDRL3 variant 2 11QQTYGTYAGSFFYS CDRL3 variant 3 12 QHTYGTYAGSFFYS CDRL3 valiant 4 13QASHYPLT CDRL3 variant 5 599 QQWSHYPLT CDRH1 14 GFSLSTYYMS CDRH1 variant1 15 GYTFTSNAIT CDRH1 variant 2 600 GYTFTSNWIT CDRH2 16 IIYPSGSTYCASWAKGCDRH2 variant 1 17 IIYPSGSTYSASWAKG CDRH2 variant 2 18 DISPGSGSTTNNEKFKSCDRH2 variant 3 601 DIYPGSGSTTNNEKFKS CDRH3 19 PDNDGTSGYLSGFGL CDRH3variant 1 20 PDNEGTSGYLSGFGL CDRH3 variant 2 21 LRGFYDYFDF CDRH3 variant3 602 LRGYYDYFDF

In one aspect, provided is a purified anti-aP2 monoclonal antibody orantigen binding fragment comprising a light chain wherein the variabledomain comprises one, two, or three CDRs independently selected fromCDRL1 (QASEDISRYLV) (Seq. ID No. 7), CDRL1 variant 1 (SVSSSISSSNLH)(Seq. ID No. 597), CDRL2 (KASTLAS) (Seq. ID No. 8), CDRL2 variant 1(GTSNLAS) (Seq. ID No. 598), CDRL3 (QCTYGTYAGSFFYS) (Seq. ID. No. 9),CDRL3 variant 1 (QATYGTYAGSFFYS) (Seq. ID No. 10), CDRL3 variant 2(QQTYGTYAGSFFYS) (Seq. ID No. 11), CDRL3 variant 3 (QHTYGTYAGSFFYS)(Seq. ID No. 12), CDRL3 variant 4 (QQASHYPLT) (Seq. ID No. 13), or CDRL3variant 5 (QQWSHYPLT) (Seq. ID No. 599). In one embodiment, providedherein is an antibody or antigen binding agent comprising a light chainvariable region comprising CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8),and CDRL3 (Seq. ID No. 9). In one embodiment, provided herein is anantibody or antigen binding agent comprising a light chain variableregion comprising CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8), andCDRL3 variant 1 (Seq. ID No. 10). In one embodiment, provided herein isan antibody or antigen binding agent comprising a light chain variableregion comprising CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8), andCDRL3 variant 2 (Seq. ID No. 11). In one embodiment, provided herein isan antibody or antigen binding agent comprising a light chain variableregion comprising CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8), andCDRL3 variant 3 (Seq. ID No. 12).

In one embodiment, provided herein is an antibody or antigen bindingagent comprising a light chain variable region comprising CDRL3 variant4 (Seq. ID No. 13), wherein the antibody has a KD of about ≧10⁻⁷ M. Inone embodiment, provided herein is an antibody or antigen binding agentcomprising a light chain variable region comprising CDRL1 variant 1(Seq. ID No. 597), CDRL2 variant 1 (Seq. ID No. 598), and CDRL3 variant4 (Seq. ID No. 13). In one embodiment, provided herein is an antibody orantigen binding agent comprising a light chain variable regioncomprising CDRL3 variant 4 (Seq. ID No. 13) and a heavy chain variableregion comprising CDHR1 variant 1 (GYTFTSNAIT) (Seq. ID No. 15), CDRH2variant 2 (DISPGSGSTTNNEKFKS) (Seq. ID No. 18), and, in one embodiment,CDRH3 variant 2 (LRGFYDYFDF) (Seq. ID No. 21).

In one embodiment, the antibody or antigen binding agent comprises one,two, or three CDRs selected from CDRL1 (Seq. ID No. 7), CDRL2 (Seq. IDNo. 8), CDRL3 (Seq. ID No. 9), CDRL3 variant 1 (Seq. ID No. 10), CDRL3variant 2 (Seq. ID No. 11), CDRL3 variant 3 (Seq. ID No. 12), and CDRL3variant 4 (Seq. ID No. 13), and has a KD of about ≧10⁻⁷ M. In oneembodiment, the CDR sequences identified above are grafted into a humanimmunoglobulin framework. In one embodiment, the human immunoglobulinframework is further modified or altered, for example within the Vernierzone, to maintain the binding affinity specificity of the grafted CDRregion.

In one embodiment, a purified anti-aP2 monoclonal antibody or antigenbinding agent is provided comprising a light chain wherein the variabledomain comprises one, two, or three CDRs independently selected from anamino acid sequence that is at least 80%, 85%, 90%, or 95% homologouswith CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8), CDRL3 (Seq. ID No.9), CDRL3 variant 1 (Seq. ID No. 10), CDRL3 variant 2 (Seq. ID No. 11),CDRL3 variant 3 (Seq. ID No. 12), or CDRL3 variant 4 (Seq. ID No. 13).In one embodiment, the antibody or antigen binding agent has a KD ofabout ≧10⁻⁷ M. In one embodiment, the CDR sequences identified above aregrafted into a human immunoglobulin framework. In one embodiment, thehuman immunoglobulin framework is further modified or altered, forexample within the Vernier zone, to maintain the binding affinityspecificity of the grafted CDR region. In one embodiment, a purifiedanti-aP2 monoclonal antibody or antigen binding agent is providedcomprising a light chain wherein the variable domain comprises one, two,or three CDRs independently selected from an amino acid sequence thathas one or more (for example, 1, 2, 3, or 4) amino acid substitutions,additions, or deletions as compared with CDRL1 (Seq. ID No. 7), CDRL2(Seq. ID No. 8), CDRL3 (Seq. ID No. 9), CDRL3 variant 1 (Seq. ID No.10), CDRL3 variant 2 (Seq. ID No. 11), CDRL3 variant 3 (Seq. ID No. 12),or CDRL3 variant 4 (Seq. ID No. 13).

In one aspect, provided is a purified anti-aP2 monoclonal antibody orantigen binding agent comprising a light chain wherein the variabledomain comprises one, two, or three CDRs selected from CDRL1 (Seq. IDNo. 7), CDRL2 (Seq. ID No. 8), CDRL3 (Seq. ID No. 9), CDRL3 variant 1(Seq. ID No. 10), CDRL3 variant 2 (Seq. ID No. 11), CDRL3 variant 3(Seq. ID No. 12), or CDRL3 variant 4 (Seq. ID No. 13), and one, two, orthree CDRs selected from CDRH1 (GFSLSTYYMS) (Seq. ID NO. 14), CDRH1variant 1 (Seq. ID No. 15), CDRH1 variant 2 (GYTFTSNWIT) (Seq. ID No.600), CDRH2 (IIYPSGSTYCASWAKG) (Seq. ID No. 16), CDRH2 variant 1(IIYPSGSTYSASWAKG) (Seq. ID No. 17), CDRH2 variant 2 (Seq. ID No. 18),CDRH2 variant 3 (DIYPGSGSTTNNEKFKS) (Seq. ID No. 601), CDHR3(PDNDGTSGYLSGFGL) (Seq. ID No. 19), CDRH3 variant 1 (PDNEGTSGYLSGFGL)(Seq. ID No. 20), CDRH3 variant 2 (Seq. ID No. 21), or CDRH3 variant 3(LRGYYDYFDFW) (Seq. ID No. 602). In one embodiment, provided herein isan antibody or antigen binding agent comprising a heavy chain variableregion comprising CDRH1 variant 1 (Seq. ID No. 15), CDRH2 variant 2(Seq. ID No. 18), and CDRH3 variant 3 (Seq. ID No. 602). In oneembodiment, provided herein is an antibody or antigen binding agentcomprising a heavy chain variable region comprising CDRH1 variant 1(Seq. ID No. 15), CDRH2 variant 2 (Seq. ID No. 18), and CDRH3 variant 2(Seq. ID No. 21). In one embodiment, the antibody or antigen bindingagent has a KD of about ≧10⁻⁷ M. In one embodiment, the CDR sequencesidentified above are grafted into a human immunoglobulin framework. Inone embodiment, the human immunoglobulin framework is further modifiedor altered, for example within the Vernier zone, to maintain the bindingaffinity specificity of the grafted CDR region.

In one embodiment, the antibody or antigen binding agent comprises one,two, or three CDRs selected from CDRH1 (Seq. ID NO. 14), CDRH1 variant 1(Seq. ID No. 15), CDRH2 (Seq. ID No. 16), CDRH2 variant 1 (Seq. ID No.17), CDRH2 variant 2 (Seq. ID No. 18), CDRH3 (Seq. ID No. 19), CDRH3variant 1 (Seq. ID No. 20), or CDRH3 variant 2 (Seq. ID No. 21), and hasa KD of about ≧10⁻⁷ M. In one embodiment, the antibody or antigenbinding agent comprises CDRs CDRH1 (Seq. ID No. 14), CDRH2 (Seq. ID No.16), and CDRH3 (Seq. ID No. 19). In one embodiment, the antibody orantigen binding agent comprises CDRs CDRH1 (Seq. ID No. 14), CDRH2variant 1 (Seq. ID No. 17), and CDHR3 variant 1 (Seq. ID No. 20). In oneembodiment, the antibody comprises CDRs CDRH1 variant 1 (Seq. ID No. 15)and CDRH2 variant 2 (Seq. ID No. 18). In one embodiment, the antibodycomprises CDRs CDRH1 variant 1 (Seq. ID No. 15), and CDRH2 variant 2(Seq. ID No. 18), and CDRH3 variant 2 (Seq. ID No. 21). In oneembodiment, the CDR sequences identified above are grafted into a humanimmunoglobulin framework. In one embodiment, the human immunoglobulinframework is further modified or altered, for example within the Vernierzone, to maintain the binding affinity specificity of the grafted CDRregion. In one embodiment, the antibody or antigen binding agentcomprises one, two, or three CDRs selected from an amino acid sequencethat has one or more (for example, 1, 2, 3, or 4) amino acidsubstitutions, additions, or deletions as compared to CDRH1 (Seq. ID NO.14), CDRH1 variant 1 (Seq. ID No. 15), CDRH2 (Seq. ID No. 16), CDRH2variant 1 (Seq. ID No. 17), CDRH2 variant 2 (Seq. ID No. 18), CDRH3(Seq. ID No. 19), CDRH3 variant 1 (Seq. ID No. 20), or CDRH3 variant 2(Seq. ID No. 21).

In one embodiment, a purified anti-aP2 monoclonal antibody or antigenbinding agent is provided comprising a heavy chain wherein the variabledomain comprises one, two, or three CDRs selected from an amino acidsequence that is at least 80%, 85%, 90%, or 95% homologous with CDRH1(Seq. ID No. 14), CDRH1 variant 1 (Seq. ID No. 15), CDRH2 (Seq. ID No.16), CDRH2 variant 1 (Seq. ID No. 17), CDRH2 variant 2 (Seq. ID No. 18),CDRH3 (Seq. ID No. 19), CDRH3 variant 1 (Seq. ID No. 20), or CDRH3variant 2 (Seq. ID No. 21). In one embodiment, the antibody or antigenbinding agent has a KD of about ≧10⁻⁷ M. In one embodiment, the CDRsequences identified above are grafted into a human immunoglobulinframework. In one embodiment, the human immunoglobulin framework isfurther modified or altered, for example within the Vernier zone, tomaintain the binding affinity specificity of the grafted CDR region.

CDRs can be altered or modified to provide for improved bindingaffinity, minimize loss of binding affinity when grafted into adifferent backbone, or to decrease unwanted interactions between the CDRand the hybrid framework as described further below.

Humanized Antibodies and Antigen Binding Agents

In one aspect of the present invention, provided herein are humanizedanti-aP2 monoclonal antibodies and antigen binding agents. Humanizedantibodies are antibodies wherein the heavy and/or light chain containsone or more CDRs (including, if desired, one or more modified CDRs) froma donor antibody (e.g. a non-human antibody such as a murine or rabbitmonoclonal antibody) grafted into a heavy and/or light chain variableregion framework of an acceptor antibody (e.g. a human antibody). For areview, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998.

In one embodiment, rather than the entire CDR being transferred, onlyone or more of the specificity determining residues from any one of theCDRs described herein above are transferred to the human antibodyframework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34).In one embodiment only the specificity determining residues from one ormore of the CDRs described herein are transferred to the human antibodyframework. In another embodiment only the specificity determiningresidues from each of the CDRs described herein are transferred to thehuman antibody framework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, rabbit, primate and human frameworkregions.

Suitably, the humanized antibody according to the present invention hasa variable domain comprising human acceptor framework regions as well asone or more of the CDRs provided specifically herein. Thus, provided inone embodiment is a humanized monoclonal antibody which binds human aP2wherein the variable domain comprises human acceptor framework regionsand non-human donor CDRs.

Construction of CDR-grafted antibodies is generally described inEuropean Patent Application EP-A-0239400, which discloses a process inwhich the CDRs of a mouse monoclonal antibody are grafted onto theframework regions of the variable domains of a human immunoglobulin bysite directed mutagenesis using long oligonucleotides, and isincorporated herein. The CDRs determine the antigen binding specificityof antibodies and are relatively short peptide sequences carried on theframework regions of the variable domains.

The earliest work on humanizing monoclonal antibodies by CDR-graftingwas carried out on monoclonal antibodies recognizing synthetic antigens,such as NP. However, examples in which a mouse monoclonal antibodyrecognizing lysozyme and a rat monoclonal antibody recognizing anantigen on human T-cells were humanized by CDR-grafting have beendescribed by Verhoeyen et al. (Science, 239, 1534-1536, 1988) andRiechmann et al (Nature, 332, 323-324, 1988), respectively. Antibodyhumanization is achieved by grafting CDRs of a non-human antibody, suchas a mouse, rat, goat, or rabbit antibody, onto a “similar” humanframework (acceptor) and selecting minimal number of key frameworkresidues (back-mutations) that are manually selected from the donormonoclonal antibody and incorporated into human acceptor framework inorder to maintain the original CDR conformation. Such methods are knownin the art, and include those described in Jones et al., Nature 321:522(1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J.Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, whichare incorporated herein.

The human variable heavy and light chain germline subfamilyclassification can be derived from the Kabat germline subgroupdesignations: VH1, VH2, VH3, VH4, VH5, VH6 or VH7 for a particular VHsequence and JH1, JH2, JH3, JH4, JH5, and JH6 for a for a particularvariable heavy joining group for framework 4; VK1, VK2, VK3, VK4, VK5 orVK6 for a particular VL kappa sequence for framework 1, 2, and 3, andJK1, JK2, JK3, JK4, or JK5 for a particular kappa joining group forframework 4; or VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, or VL10 fora particular VL lambda sequence for framework 1, 2, and 3, and JL1, JL2,JL3, or JL7 for a particular lambda joining group for framework 4.

In one embodiment, the general framework of the light chain contemplatedherein comprises the structures selected fromFR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4 andFR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4-CL, and variations thereof, whereinthe CDR regions are selected from at least one variable light chain CDRselected from Seq. ID Nos. 7-13, the framework regions are selected fromeither an immunoglobulin kappa light chain variable framework region,for example as provided in Table 4 (Seq. ID Nos. 25-149), or animmunoglobulin lambda light chain variable framework region, for exampleas provided in Table 5 (Seq. ID Nos. 150-246), and an immunoglobulinlight chain constant region from either a kappa light chain constantregion (Seq. ID No. 247) when the framework region is a kappa lightchain variable framework region, or a lambda light chain constant region(Seq. ID No. 248) when the framework region is a lambda light chainvariable framework region.

In one embodiment, the general framework of the heavy chain regionscontemplated herein comprises the structures selected fromFR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4,FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1,FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1-Hinge-CH2 for IgG, IgD, and IgAimmunoglobulin classes and FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1-CH2 forIgM and IgE immunoglobulin classes,FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1-Hinge-CH2-CH3 for IgG, IgD, andIgA immunoglobulin classes,FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1-CH2-CH3 for IgM and IgEimmunoglobulin classes, andFR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4-CH1-CH2-CH3-CH4 for IgM and IgEimmunoglobulin classes, and variations thereof, wherein the CDR regionsare selected from at least one variable heavy chain CDR selected fromSeq. ID Nos. 14-21, and the framework regions are selected from theheavy chain variable framework regions described in Table 7 (Seq. IDNos. 249-407), and the heavy chain constant regions are selected from,for example, those provided in Table 8 (Seq. ID Nos. 408-443). IgA andIgM classes can further comprise a joining polypeptide (Seq. ID No. 444)provided in Table 9 that serves to link two monomer units of IgM or IgAtogether, respectively. In the case of IgM, the J chain-joined dimer isa nucleating unit for the IgM pentamer, and in the case of IgA itinduces larger polymers.

The constant region domains of the antibody molecule of the presentinvention, if present, may be selected having regard to the proposedfunction of the antibody molecule, and in particular the effectorfunctions which may be required. For example, the constant regiondomains may be human IgA, IgD, IgE, IgG or IgM domains. In particularembodiments, human IgG constant region domains may be used, especiallyof the IgG1 and IgG3 isotypes when the antibody molecule is intended fortherapeutic uses and antibody effector functions are required.Alternatively, IgG2 and IgG4 isotypes may be used when the antibodymolecule is intended for therapeutic purposes and antibody effectorfunctions are not required. It will be appreciated that sequencevariants of these constant region domains may also be used. For exampleIgG4 molecules in which the serine at position 241 has been changed toproline as described in Angal et al., Molecular Immunology, 1993, 30(1), 105-108 may be used. It will also be understood by one skilled inthe art that antibodies may undergo a variety of posttranslationalmodifications. The type and extent of these modifications often dependson the host cell line used to express the antibody as well as theculture conditions. Such modifications may include variations inglycosylation, methionine oxidation, diketopiperazine formation,aspartate isomerization and asparagine deamidation. A frequentmodification is the loss of a carboxy-terminal basic residue (such aslysine or arginine) due to the action of carboxypeptidases (as describedin Harris, R J. Journal of Chromatography 705:129-134, 1995).Accordingly, the C-terminal lysine of the antibody heavy chain may beabsent.

In one embodiment, the anti-aP2 monoclonal antibody comprises at leastone light chain CDR selected from CDRL1 (Seq. ID No. 7), CDRL2 (Seq. IDNo. 8), CDRL3 (Seq. ID No. 9), or CDRL3 variant 1 (Seq. ID No. 10),CDRL3 variant 2 (Seq. ID No. 11), CDRL3 variant 3 (Seq. ID NO. 12), andCDRL3 variant 4 (Seq. ID No. 13), and/or at least one heavy chain CDRselected from CDRH1 (Seq. ID No. 14), CDRH1 variant 1 (Seq. ID No. 15),CDRH2 (Seq. ID No. 16), CDRH2 variant 1 (Seq. ID No. 17), CDRH2 variant2 (Seq. ID No. 18), CDRH3 (Seq. ID No. 19), CDRH3 variant 1 (Seq. ID No20), or CDRH3 variant 2 (Seq. ID No. 21), or a combination or variantthereof, wherein the CDR is grafted into a human light or heavy chainvariable framework, respectively.

In one embodiment, the anti-aP2 monoclonal antibody comprises one, two,or three light chain CDRs selected from CDRL1 (Seq. ID No. 7), CDRL2(Seq. ID No. 8), CDRL3 (Seq. ID No. 9), CDRL3 variant 1 (Seq. ID No.10), CDRL3 variant 2 (Seq. ID No. 11), CDRL3 variant 3 (Seq. ID NO. 12),and CDRL3 variant 4 (Seq. ID No. 13), or a combination or variantthereof, grafted into a human acceptor light chain framework. In oneembodiment, the anti-aP2 monoclonal antibody comprises a variable lightchain comprising CDRL1 (Seq. ID No. 7), CDRL2 (Seq. ID No. 8), and CDRL3(Seq. ID No. 9) or CDRL3 variant 1 (Seq. ID No. 10) or CDRL3 variant 2(Seq. ID No. 11) or CDRL3 variant 3 (Seq. ID No. 12), or a combinationor variant thereof, grafted into a human acceptor light chain framework.In one embodiment, the human acceptor light chain framework is derivedfrom an amino acid sequence encoded by a human IGKV (VL kappa) gene forframework 1, 2, and 3, and an IGKJ gene for framework 4. In oneembodiment, the human acceptor light chain framework is derived from anamino acid sequence encoded by a human IGLV (VL lambda) gene forframework 1, 2, and 3, and an IGLJ gene for framework 4. Non-limitingexamples of human light chain IGKV and IGKJ acceptor framework regionsare provided, for example, in Table 4, and non-limiting examples ofhuman light chain IGLV and IGLJ acceptor framework regions are provide,for example, Table 5.

TABLE 4 Human IGKV and IGKJ Framework Regions Variable Light κ Chain FRRegion Seq. ID No. Sequence O12 FR1 25 DIQMTQSPSSLSASVGDRVTITC O12 FR226 WYQQKPGKAPKLLIY O12 FR3 27 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC O2 FR1 28DIQMTQSPSSLSASVGDRVTITC O2 FR2 29 WYQQKPGKAPKLLIY O2 FR3 30GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC O18 FR1 31 DIQMTQSPSSLSASVGDRVTITC O18FR2 32 WYQQKPGKAPKLLIY O18 FR3 33 GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC O8FR1 34 DIQMTQSPSSLSASVGDRVTITC O8 FR2 35 WYQQKPGKAPKLLIY O8 FR3 36GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC A20 FR1 37 DIQMTQSPSSLSASVGDRVTITC A20FR2 38 WYQQKPGKVPKLLIY A20 FR3 39 GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC A30FR1 40 DIQMTQSPSSLSASVGDRVTITC A30 FR2 41 WYQQKPGKAPKRLIY A30 FR3 42GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC L14 FR1 43 NIQMTQSPSAMSASVGDRVTITC L14FR2 44 WFQQKPGKVPKHLIY L14 FR3 45 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC L1FR1 46 DIQMTQSPSSLSASVGDRVTITC L1 FR2 47 WFQQKPGKAPKSLIY L1 FR3 48GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L15 FR1 49 DIQMTQSPSSLSASVGDRVTITC L15FR2 50 WYQQKPEKAPKSLIY L15 FR3 51 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L4FR1 52 AIQLTQSPSSLSASVGDRVTITC L4 FR2 53 WYQQKPGKAPKLLIY L4 FR3 54GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L18 FR1 55 AIQLTQSPSSLSASVGDRVTITC L18FR2 56 WYQQKPGKAPKLLIY L18 FR3 57 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L5FR1 58 DIQMTQSPSSVSASVGDRVTITC L5 FR2 59 WYQQKPGKAPKLLIY L5 FR3 60GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L19 FR1 61 DIQMTQSPSSVSASVGDRVTITC L19FR2 62 WYQQKPGKAPKLLIY L19 FR3 63 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L8FR1 64 DIQLTQSPSFLSASVGDRVTITC L8 FR2 65 WYQQKPGKAPKLLIY L8 FR3 66GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC L23 FR1 67 AIRMTQSPFSLSASVGDRVTITC L23FR2 68 WYQQKPAKAPKLFIY L23 FR3 69 GVPSRFSGSGSGTDYTLTTSSLQPEDFATYYC L9FR1 70 AIRMTQSPSSFSASTGDRVTITC L9 FR2 71 WYQQKPGKAPKLLIY L9 FR3 72GVPSRFSGSGSGTDFTLTTSCLQSEDFATYYC L24 FR1 73 VIWMTQSPSLLSASTGDRVTISC L24FR2 74 WYQQKPGKAPELLTY L24 FR3 75 GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC L11FR1 76 AIQMTQSPSSLSASVGDRVTITC L11 FR2 77 WYQQKPGKAPKLLIY L11 FR3 78GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L12 FR1 79 DIQMTQSPSTLSASVGDRVTITC L12FR2 80 WYQQKPGKAPKLLIY L12 FR3 81 GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC O11FR1 82 DIVMTQTPLSLPVTPGEPASISC O11 FR2 83 WYLQKPGQSPQLLIY O11 FR3 84GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC O1 FR1 85 DIVMTQTPLSLPVTPGEPASISC O1FR2 86 WYLQKPGQSPQLLIY O1 FR3 87 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A17FR1 88 DVVMTQSPLSLPVTLGQPASISC A17 FR2 89 WFQQRPGQSPRRLIY A17 FR3 90GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A1 FR1 91 DVVMTQSPLSLPVTLGQPASISC A1FR2 92 WFQQRPGQSPRRLIY A1 FR3 93 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A18FR1 94 DIVMTQTPLSLSVTPGQPASISC A18 FR2 95 WYLQKPGQSPQLLIY A18 FR3 96GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A2 FR1 97 DIVMTQTPLSLSVTPGQPASISC A2FR2 98 WYLQKPGQPPQLLIY A2 FR3 99 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A19FR1 100 DIVMTQSPLSLPVTPGEPASISC A19 FR2 101 WYLQKPGQSPQLLIY A19 FR3 102GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A3 FR1 103 DIVMTQSPLSLPVTPGEPASISC A3FR2 104 WYLQKPGQSPQLLIY A3 FR3 105 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A23FR1 106 DIVMTQTPLSSPVTLGQPASISC A23 FR2 107 WLQQRPGQPPRLLIY A23 FR3 108GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC A27 FR1 109 EIVLTQSPGTLSLSPGERATLSE A27FR2 110 WYQQKPGQAPRLLIY A27 FR3 111 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC A11FR1 112 EIVLTQSPATLSLSPGERATLSC A11 FR2 113 WYQQKPGLAPRLLIY A11 FR3 114GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC L2 FR1 115 EIVMTQSPATLSVSPGERATLSC L2FR2 116 WYQQKPGQAPRLLIY L2 FR3 117 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC L16FR1 118 EIVMTQSPATLSVSPGERATLSC L16 FR2 119 WYQQKPGQAPRLLIY L16 FR3 120GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC L6 FR1 121 EIVLTQSPATLSLSPGERATLSC L6FR2 122 WYQQKPGQAPRLLIY L6 FR3 123 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC L20FR1 124 EIVLTQSPATLSLSPGERATLSC L20 FR2 125 WYQQKPGQAPRLLIY L20 FR3 126GIPARFSGSGPGTDFTLTISSLEPEDFAVYYC L25 FR1 127 EIVMTQSPATLSLSPGERATLSC L25FR2 128 WYQQKPGQAPRLLIY L25 FR3 129 GIPARFSGSGSGTDFTLTISSLQPEDFAVYYC B3FR1 130 DIVMTQSPDSLAVSLGERATINC B3 FR2 131 WYQQKPGQPPKLLIY B3 FR3 132GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC B2 FR1 133 ETTLTQSPAFMSATPGDKVNISC B2FR2 134 WYQQKPGEAAIFIIQ B2 FR3 135 GIPPRFSGSGYGTDFTLTINNIESEDAAYYFC A26FR1 136 EIVLTQSPDFQSVTPKEKVTITC A26 FR2 137 WYQQKPDQSPKLLIK A26 FR3 138GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC A10 FR1 139 EIVLTQSPDFQSVTPKEKVTITC A10FR2 140 WYQQKPDQSPKLLIK A10 FR3 141 GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC A14FR1 142 DvvMTQSPAFLSVTPGEKVTITC A14 FR2 143 WYQQKPDQAPKLLIK A14 FR3 144GVPSRFSGSGSGTDFTFTISSLEAEDAATYYC JK1 FR4 145 FGQGTKVEIK JK2 FR4 146FGQGTKLEIK JK3 FR4 147 FGPGTKVDIK JK4 FR4 148 FGGGTKVEIK JK5 FR4 149FGQGTRLEIK

TABLE 5 Human IGLV and IGLJ Framework Regions Variable Light λ Chain FRRegion Seq. ID No. Sequence 1a FR1 150 QSVLTQPPSVSEAPRQRVTISC 1a FR2 151WYQQLPGKAPKLLIY 1a FR3 152 GVSDRFSGSKSG-TSASLAISGLQSEDEADYYC 1e FR1 153QSVLTQPPSVSGAPGQRVTISC 1e FR2 154 WYQQLPGTAPKLLIY 1e FR3 155GVPDRFSGSKSG-TSASLAITGLQAEDEADYYC 1c FR1 156 QSVLTQPPSASGTPGQRVTISC 1cFR2 157 WYQQLPGTAPKLLIY 1c FR3 158 GVPDRFSGSKSG-TSASLAISGLQSEDEADYYC 1gFR1 159 QSVLTQPPSASGTPGQRVTISC 1g FR2 160 WYQQLPGTAPKLLIY 1g FR3 161GVPDRFSGSKSG-TSASLAISGLRSEDEADYYC 1b FR1 162 QSVLTQPPSVSAAPGQKVTISC 1bFR2 163 WYQQLPGTAPKLLIY 1b FR3 164 GIPDRFSGSKSG-TSATLGITGLQTGDEADYYC 2cFR1 165 QSALTQPPSASGSPGQSVTISC 2c FR2 166 WYQQHPGKAPKLMIY 2c FR3 167GVPDRFSGSKSG-NTASLTVSGLQAEDEADYYC 2e FR1 168 QSALTQPRSVSGSPGQSVTISC 2eFR2 169 WYQQHPGKAPKLMIY 2e FR3 170 GVPDRFSGSKSG-NTASLTISGLQAEDEADYYC 2a2FR1 171 QSALTQPASVSGSPGQSITISC 2a2 FR2 172 WYQQHPGKAPKLMIY 2a2 FR3 173GVSNRFSGSKSG-NTASLTISGLQAEDEADYYC 2d FR1 174 QSALTQPPSVSGSPGQSVTISC 2dFR2 175 WYQQPPGTAPKLMIY 2d FR3 176 GVPDRFSGSKSG-NTASLTISGLQAEDEADYYC 2b2FR1 177 QSALTQPASVSGSPGQSITISC 2b2 FR2 178 WYQQHPGKAPKLMIY 2b2 FR3 179GVSNRFSGSKSG-NTASLTISGLQAEDEADYYC 3r FR1 180 SYELTQPPSVSVSPGQTASITC 3rFR2 181 WYQQKPGQSPVLVIY 3r FR3 182 GIPERFSGSNSG-NTATLTISGTQAMDEADYYC 3jFR1 183 SYELTQPLSVSVALGQTARITC 3j FR2 184 WYQQKPGQAPVLVIY 3j FR3 185GIPERFSGSNSG-NTATLTISRAQAGDEADYYC 3p FR1 186 SYELTQPPSVSVSPGQTARITC 3pFR2 187 WYQQKSGQAPVLVIY 3p FR3 188 GIPERFSGSSSG-TMATLTISGAQVEDEADYYC 3aFR1 189 SYELTQPPSVSVSLGQMARITC 3a FR2 190 WYQQKPGQFPVLVIY 3a FR3 191GIPERFSGSSSG-TIVTLTISGVQAEDEADYYC 3l FR1 192 SSELTQDPAVSVALGQTVRITC 3lFR2 193 WYQQKPGQAPVLVIY 3l FR3 194 GIPDRFSGSSSG-NTASLTITGAQAEDEADYYC 3hFR1 195 SYVLTQPPSVSVAPGKTARITC 3h FR2 196 WYQQKPGQAPVLVIY 3h FR3 197GIPERFSGSNSG-NTATLTISRVEAGDEADYYC 3e FR1 198 SYELTQLPSVSVSPGQTARITC 3eFR2 199 WYQQKPGQAPELVIY 3e FR3 200 GIPERFSGSTSG-NTTTLTISRVLTEDEADYYC 3mFR1 201 SYELMQPPSVSVSPGQTARITC 3m FR2 202 WYQQKPGQAPVLVIY 3m FR3 203GIPERFSGSSSG-TTVTLTISGVQAEDEADYYC 2-19 FR1 204 SYELTQPSSVSVSPGQTARITC2-19 FR2 205 WFQQKPGQAPVLVIY 2-19 FR3 206GIPERFSGSSSG-TTVTLTISGAQVEDEADYYC 4c FR1 207 LPVLTQPPSASALLGASIKLTC 4cFR2 208 WYQQRPGRSPQYIMK 4c FR3 209 GIPDRFMGSSSG-ADRYLTFSNLQSDDENEYHC 4aFR1 210 QPVLTQSSSASASLGSSVKLTC 4a FR2 211 WHQQQPGKAPRYLMK 4a FR3 212GVPDRFSGSSSG-ADRYLTISNLQLEDEADYYC 4b FR1 213 QLVLTQSPSASASLGASVKLTC 4hFR2 214 WHQQQPEKGPRYLMK 4b FR3 215 GIPDRFSGSSSG-AERYLTISSLQSEDEADYYC 5eFR1 216 QPVLTQPPSSSASPGESARLTC 5e FR2 217 WYQQKPGSPPRYLLY 5e FR3 218GVPSRFSGSKDASANTGILLISGLQSEDEADYYC 5c FR1 219 QAVLTQPASLSASPGASASLTC 5cFR2 220 WYQQKPGSPPQYLLR 5c FR3 221 GVPSRFSGSKDASANAGILLISGLQSEDEADYYC 5bFR1 222 QPVLTQPSSHSASSGASVRLTC 5b FR2 223 WYQQKPGNPPRYLLY 5b FR3 224GVPSRFSGSNDASANAGILRISGLQPEDEADYYC 6a FR1 225 NFMLTQPHSVSESPGKTVTISC 6aFR2 226 WYQQRPGSSPTTVIY 6a FR3 227 GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC 7aFR1 228 QTVVTQEPSLTVSPGGTVTLTC 7a FR2 229 WFQQKPGQAPRALIY 7a FR3 230WTPARFSGSLLG-GKAALTLSGVQPEDEAEYYC 7b FR1 231 QAVVTQEPSLTVSPGGTVTLTC 7bFR2 232 WFQQKPGQAPRTLIY 7b FR3 233 WTPARFSGSLLG-GKAALTLSGAQPEDEAEYYC 8aFR1 234 QTVVTQEPSFSVSPGGTVTLTC 8a FR2 235 WYQQTPGQAPRTLIY 8a FR3 236GVPDRFSGSILG-NKAALTITGAQADDESDYYC 9a FR1 237 QPVLTQPPSASASLGASVTLTC 9aFR2 238 WYQQRPGKGPRFVMR 9a FR3 239 GIPDRFSVLGSG-LNRYLTIKNIQEEDESDYHC 10aFR1 240 QAGLTQPPSVSKGLRQTATLTC 10a FR2 241 WLQQHQGHPPKLLSY 10a FR3 242GISERLSASRSG-NTASLTITGLQPEDEADYYC JL1 FR4 243 FGTGTKVTVL JL2 FR4 244FGGGTKLTVL JL3 FR4 245 FGGGTKLTVL JL7 FR4 246 FGGGTQLTVL

The immunoglobulin constant light chain region for use in the presentinvention is determined by the variable light chain the CDRs are graftedinto. For example, if the variable light chain FR regions are derivedfrom the immunoglobulin kappa light chain variable region, then aconstant light chain region from an immunoglobulin kappa light chainconstant region (IGKC) can be used to produce a light chain VL-CL chain.An IGKC that may be used in the present invention includes Seq. ID No.247 in Table 6 below. Conversely, when the framework region isimmunoglobulin lambda light chain variable region, then animmunoglobulin lambda light chain constant region (IGLC) may be used toproduce a lambda VL-CL light chain. An immunoglobulin lambda light chainconstant region that may be used in the present invention includes (Seq.ID No. 248) in Table 6 below, and allelic variants thereof, which aregenerally known in the art, for example as identified in OMIM entry147200 for IGKC variants and 147220 for IGLC variants.

TABLE 6 Sequence of Human Immunoglobulin Light Chain Constant Regions IgLight Chain Constant Region Seq. ID No. Sequence Ig Kappa Constant 247TVAAPSVFIFPPSDEQLKSGTASVVCLLNN Region (IGKC)FYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda 248 QPKAAPSVTLFPPSSEELQANKATLVCLIS ConstantRegion (IGLC) DFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS

In one embodiment, the anti-aP2 monoclonal antibody has at least oneheavy chain CDR selected from CDRH1 (Seq. ID No. 14), CDRH1 variant 1(Seq. ID No. 15), CDRH2 (Seq. ID No. 16), CDRH2 variant 1 (Seq. ID No.17), CDRH2 variant 2 (Seq. ID No. 18), CDRH3 (Seq. ID No 19), CDRH3variant 1 (Seq. ID No. 20), CHRH3 variant 2 (Seq. ID No. 21), or acombination or variant thereof, grafted into a human acceptor heavychain framework. In one embodiment, the anti-aP2 monoclonal antibodycomprises CDRs CDRH1 (Seq. ID No. 14), CDRH2 (Seq. ID No. 16), CDRH3(Seq. ID No. 19), or variant thereof, grafted into a human acceptorheavy chain framework. In one embodiment, the anti-aP2 monoclonalantibody comprises CDRs CDRH1 (Seq. ID No. 14), CDRH2 variant 1 (Seq. IDNo. 17), or CDRH3 variant 1 (Seq. ID No 20), or a variant thereof,grafted into a human acceptor heavy chain framework. In one embodiment,the anti-aP2 monoclonal antibody comprises CDRs CDRH1 variant 1 (Seq. IDNo. 15) and CDRH2 variant 2 (Seq. ID No. 18), or a variant thereof,grafted into a human acceptor heavy chain framework. In one embodiment,the anti-aP2 monoclonal antibody comprises CDRs CDRH1 variant 1 (Seq. IDNo. 15), CDRH2 variant 2 (Seq. ID No. 18), or CDRH3 variant 2 (Seq. IDNo 21), or a variant thereof, grafted into a human acceptor heavy chainframework. In one embodiment, the human acceptor heavy chain frameworkis derived from an amino acid sequence encoded by a human IGHV gene forframework 1, 2, and 3, and an IGHJ gene for framework 4. Non-limitingexamples of human heavy chain IGHV and IGHJ acceptor framework regionsare provided, for example, in Table 7.

TABLE 7 Sequences of Human Immunoglobulin Heavy Chain Variable RegionsHeavy Chain Variable Seq. FR ID Regions No. Sequence 1-02 FR1 249QVQLVQSGAEVKKPGASVKVSCKAS 1-02 FR2 250 WVRQAPGQGLEWMG 1-02 FR3 251RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 1-03 FR1 252 QVQLVQSGAEVKKPGASVKVSCKAS1-03 FR2 253 WVRQAPGQRLEWMG 1-03 FR3 254RVTITRDTSASTAYMELSSLRSEDTAVYYCAR 1-08 FR1 255 QVQLVQSGAEVKKPGASVKVSCKAS1-08 FR2 256 WVRQATGQGLEWMG 1-08 FR3 257RVTMTRNTSISTAYMELSSLRSEDTAVYYCAR 1-18 FR1 258 QVQLVQSGAEVKKPGASVKVSCKAS1-18 FR2 259 WVRQAPGQGLEWMG 1-18 FR3 260RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR 1-24 FR1 261 QVQLVQSGAEVKKPGASVKVSCKVS1-24 FR2 262 WVRQAPGKGLEWMG 1-24 FR3 263RVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT 1-45 FR1 264 QMQLVQSGAEVKKTGSSVKVSCKAS1-45 FR2 265 WVRQAPGQALEWMG 1-45 FR3 266RVTITRDRSMSTAYMELSSLRSEDTAMYYCAR 1-46 FR1 267 QVQLVQSGAEVKKPGASVKVSCKAS1-46 FR2 268 WVRQAPGQGLEWMG 1-46 FR3 269RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 1-58 FR1 270 QMQLVQSGPEVKKPGTSVKVSCKAS1-58 FR2 271 WVRQARGQRLEWIG 1-58 FR3 272RVTITRDMSTSTAYMELSSLRSEDTAVYYCAA 1-69 FR1 273 QVQLVQSGAEVKKPGSSVKVSCKAS1-69 FR2 274 WVRQAPGQGLEWMG 1-69 FR3 275RVTITADESTSTAYMELSSLRSEDTAVYYCAR 1-e FR1 276 QVQLVQSGAEVKKPGSSVKVSCKAS1-e FR2 277 WVRQAPGQGLEWMG 1-e FR3 278 RVTITADKSTSTAYMELSSLRSEDTAVYYCAR1-f FR1 279 EVQLVQSGAEVKKPGATVKISCKVS 1-f FR2 280 WVQQAPGKGLEWMG 1-f FR3281 RVTITADTSTDTAYMELSSLRSEDTAVYYCAT 2-05 FR1 282QITLKESGPTLVKPTQTLTLTCTFS 2-05 FR2 283 WIRQPPGKALEWLA 2-05 FR3 284RLTITKDTSKNQVVLTMTNMDPVDTATYYCAHR 2-26 FR1 285 QVTLKESGPVLVKPTETLTLTCTVS2-26 FR2 286 WIRQPPGKALEWLA 2-26 FR3 287RLTISKDTSKSQVVLTMTNMDPVDTATYYCARI 2-70 FR1 288 QVTLKESGPALVKPTQTLTLTCTFS2-70 FR2 289 WIRQPPGKALEWLA 2-70 FR3 290RLTISKDTSKNQVVLTMTNMDPVDTATYYCARI 3-07 FR1 291 EVQLVESGGGLVQPGGSLRLSCAAS3-07 FR2 292 WVRQAPGKGLEWVA 3-07 FR3 293RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 3-09 FR1 294 EVQLVESGGGLVQPGRSLRLSCAAS3-09 FR2 295 WVRQAPGKGLEWVS 3-09 FR3 296RFTISRDNAKNSLYLQMNSLRAEDTALYYCAKD 3-11 FR1 297 QVQLVESGGGLVKPGGSLRLSCAAS3-11 FR2 298 WIRQAPGKGLEWVS 3-11 FR3 299RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 3-13 FR1 300 EVQLVESGGGLVQPGGSLRLSCAAS3-13 FR2 301 WVRQATGKGLEWVS 3-13 FR3 302RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR 3-15 FR1 303 EVQLVESGGGLVKPGGSLRLSCAAS3-15 FR2 304 WVRQAPGKGLEWVG 3-15 FR3 305RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 3-20 FR1 306 EVQLVESGGGVVRPGGSLRLSCAAS3-20 FR2 307 WVRQAPGKGLEWVS 3-20 FR3 308RFTISRDNAKNSLYLQMNSLRAEDTALYHCAR 3-21 FR1 309 EVQLVESGGGLVKPGGSLRLSCAAS3-21 FR2 310 WVRQAPGKGLEWVS 3-21 FR3 311RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 3-23 FR1 312 EVQLLESGGGLVQPGGSLRLSCAAS3-23 FR2 313 WVRQAPGKGLEWVS 3-23 FR3 314RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 3-30 FR1 315 QVQLVESGGGVVQPGRSLRLSCAAS3-30 FR2 316 WVRQAPGKGLEWVA 3-30 FR3 317RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 3-30.3 FR1 318QVQLVESGGGVVQPGRSLRLSCAAS 3-30.3 FR2 319 WVRQAPGKGLEWVA 3-30.3 FR3 320RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 3-3 FR1 321 QVQLVESGGGVVQPGRSLRLSCAAS3-30.5 FR2 322 WVRQAPGKGLEWVA 3-30.5 FR3 323RFFISRDNSKNTLYLQMNSLRAEDTAVYYCAK 3-3 FR13 324 QVQLVESGGGVVQPGRSLRLSCAAS3-33 FR2 325 WVRQAPGKGLEWVA 3-33 FR3 326RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 3-43 FR1 327 EVQLVESGGVVVQPGGSLRLSCAAS3-43 FR2 328 WVRQAPGKGLEWVS 3-43 FR3 329RFTISRDNSKNSLYLQMNSLRTEDTALYYCAKD 3-48 FR1 330 EVQLVESGGGLVQPGGSLRLSCAAS3-48 FR2 331 WVRQAPGKGLEWVS 3-48 FR3 332RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR 3-49 FR1 333 EVQLVESGGGLVQPGRSLRLSCTAS3-49 FR2 334 WFRQAPGKGLEWVG 3-49 FR3 335RFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR 3-53 FR1 336 EVQLVETGGGLIQPGGSLRLSCAAS3-53 FR2 337 WVRQAPGKGLEWVS 3-53 FR3 338RFFISRDNSKNTLYLQMNSLRAEDTAVYYCAR 3-64 FR1 339 EVQLVESGGGLVQPGGSLRLSCAAS3-64 FR2 340 WVRQAPGKGLEYVS 3-64 FR3 341RFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR 3-66 FR1 342 EVQLVESGGGLVQPGGSLRLSCAAS3-66 FR2 343 WVRQAPGKGLEWVS 3-66 FR3 344RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 3-72 FR1 345 EVQLVESGGGLVQPGGSLRLSCAAS3-72 FR2 346 WVRQAPGKGLEWVG 3-72 FR3 347RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR 3-73 FR1 348 EVQLVESGGGLVQPGGSLKLSCAAS3-73 FR2 349 WVRQASGKGLEWVG 3-73 FR3 350RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR 3-74 FR1 351 EVQLVESGGGLVQPGGSLRLSCAAS3-74 FR2 352 WVRQAPGKGLVWVS 3-74 FR3 353RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR 3-d FR1 354 EVQLVESRGVLVQPGGSLRLSCAAS3-d FR2 355 WVRQAPGKGLEWVS 3-d FR3 356 RFTISRDNSKNTLHLQMNSLRAEDTAVYYCKK4-04 FR1 357 QVQLQESGPGLVKPSGTLSLTCAVS 4-04 FR2 358 WVRQPPGKGLEWIG 4-04FR3 359 RVTISVDKSKNQFSLKLSSVTAADTAVYYCAR 4-28 FR1 360QVQLQESGPGLVKPSDTLSLTCAVS 4-28 FR2 361 WIRQPPGKGLEWIG 4-28 FR3 362RVTMSVDTSKNQFSLKLSSVTAVDTAVYYCAR 4-30.1 FR1 363QVQLQESGPGLVKPSQTLSLTCTVS 4-30.1 FR2 364 WIRQHPGKGLEWIG 4-30.1 FR3 365RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-3 FR1 366 QLQLQESGSGLVKPSQTLSLTCAVS4-30.2 FR2 367 WIRQPPGKGLEWIG 4-30.2 FR3 368RVTISVDRSKNQFSLKLSSVTAADTAVYYCAR 4-3 FR10.4 369QVQLQESGPGLVKPSQTLSLTCTVS 4-30.4 FR2 370 WIRQPPGKGLEWIG 4-30.4 FR3 371RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-3 FR1 372 QVQLQESGPGLVKPSQTLSLTCTVS4-31 FR2 373 WIRQHPGKGLEWIG 4-31 FR3 374RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-34 FR1 375 QVQLQQWGAGLLKPSETLSLTCAVY4-34 FR2 376 WIRQPPGKGLEWIG 4-34 FR3 377RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-39 FR1 378 QLQLQESGPGLVKPSETLSLTCTVS4-39 FR2 379 WIRQPPGKGLEWIG 4-39 FR3 380RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-59 FR1 381 QVQLQESGPGLVKPSETLSLTCTVS4-59 FR2 382 WIRQPPGKGLEWIG 4-59 FR3 383RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-61 FR1 384 QVQLQESGPGLVKPSETLSLTCTVS4-61 FR2 385 WIRQPPGKGLEWIG 4-61 FR3 386RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 4-b FR1 387 QVQLQESGPGLVKPSETLSLTCAVS4-b FR2 388 WIRQPPGKGLEWIG 4-b FR3 389 RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR5-51 FR1 390 EVQLVQSGAEVKKPGESLKISCKGS 5-51 FR2 391 WVRQMPGKGLEWMG 5-51FR3 392 QVTISADKSISTAYLQWSSLKASDTAMYYCAR 5-a FR1 393EVQLVQSGAEVKKPGESLRISCKGS 5-a FR2 394 WVRQMPGKGLEWMG 5-a FR3 395HVTISADKSISTAYLQWSSLKASDTAMYYCAR 6-01 FR1 396 QVQLQQSGPGLVKPSQTLSLTCAIS6-01 FR2 397 WIRQSPSRGLEWLG 6-01 FR3 398RITINPDTSKNQFSLQLNSVTPEDTAVYYCAR 7-4.1 FR1 399 QVQLVQSGSELKKPGASVKVSCKAS7-4.1 FR2 400 WVRQAPGQGLEWMG 7-4.1 FR3 401RFVFSLDTSVSTAYLQICSLKAEDTAVYYCAR JH1 FR4 402 WGQGTLVTVSS JH2 FR4 403WGRGTLVTVSS JH3 FR4 404 WGQGTMVTVSS JH4 FR4 405 WGQGTLVTVSS JH5 FR4 406WGQGTLVTVSS JH6 FR4 407 WGQGTTVTVSS

The immunoglobulin heavy chain constant region for use in the presentinvention is determinant on the immunoglobulin class desired. Allclasses of immunoglobulins—IgG, IgD, IgA, IgM and IgE—are hereincontemplated. For example, if the desired immunoglobulin is IgG, thenthe amino acid sequence encoding the IgG heavy chain constant region(IGGH) may be used. Immunoglobulin heavy chain constant regions that maybe used in the present invention include those of IGGH, IGDH, IGAH,IGMH, and IGEH (Seq. ID Nos. 408-443) provided in Table 8 below, andallelic variants thereof, which are generally known in the art, forexample as identified in OMIM entry 147100 for IGGH1 variants, 147110for IGGH2 variants, 147120 for IGGH3 variants, 147130 for IGGH4variants, 146900 for IGAH1 variants, 147000 for IGAH2 variants, 147180for IGEH variants, 147020 for IGMH variants, 147170 for IGDH variants,all of which are incorporated by reference herein. In certainembodiment, the hinge region of a particular immunoglobulin class may beused in constructing the antibody contemplated herein. In oneembodiment, the hinge region can be derived from a natural hinge regionamino acid sequence as described in Table 8 (Seq. ID Nos. 409, 413, 417,425, 429, 433, and 437), or a variant thereof. In one embodiment, thehinge region can be synthetically generated. Further contemplated hereinare antibodies of immunoglobulin class IgA and IgM, which, in oneembodiment, may be complexed with a joining polypeptide described inTable 9, or a variant thereof.

TABLE 8 Immunoglobulin Heavy Chain Constant Region Heavy Chain ConstantRegion Seq. ID No. Sequence IGAH1 CH1 408ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNP SQDVTVPCP IGAH1 Hinge 409PSTPPTPSPSTPPTPSPS IGAH1 CH2 410CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYTPESKT PLTATLSKS IGAH1 CH3 411GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSC MVGHEALPLAFTQKTIDRLA IGAH2CH1 412 ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTN PSQDVTVPCP IGAH2 Hinge413 PPPPP IGAH2 CH2 414 CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELK TPLTANITKS IGAH2 CH3 415GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSC MVGHEALPLAFTQKTIDRLA IGDHCH1 416 APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYIVITSSQLSTPLQQWRQGEYKCVVQHTAS KSKKEIFRWP IGDH Hinge417 ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKE KEEQEERETKTP IGDH CH2418 ECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCT LNHPSLPPQRLMALREP IGDHCH3 419 AAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSH EDSRTLLNASRSLEVS IGEH CH1420 ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTCDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSS TDWVDNKTFS IGEH CH2 421VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVT YQGHTFEDSTKKCA IGEH CH3422 DSNPRGVSAYLSRPSPFTDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVT HPHLPRALMRSTTKTS IGEH CH4423 GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHE AASPSQTVQRAVSVNP IGGH1CH1 424 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVXQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV IGGH1 Hinge 425EPKSCDKTHTCPPCP IGGH1 CH2 426APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAK IGGH1CH3 427 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSP IGGH2 CH1428 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTV IGGH2 Hinge 429ERKCCVECPPCP IGGH2 CH2 430APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTK IGGH2CH3 431 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQOGNVFSCSVMHEA LHNHYTQKSLSLSP IGGH3 CH1432 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTK VDKRV IGGH3 Hinge 433ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPE PKSCDTPPPCPRCP IGGH3CH2 434 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKTK IGGH3CH3 435 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWSSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL HNRFTQKSLSLSP IGGH4 CH1436 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQSSGLYSLSSVVTWSSSLGTKTYTCNVDHKPSNTK VDKRV IGGH4 Hinge 437ESKYGPPCPSCP IGGH4 CH2 438 APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK IGGH4CH3 439 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSL IGMH CH1440 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDWQGTDEHVVCKVQH PNGNKEKNVPLP IGMH CH2 441VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFT CRVDHRGLTFQQNASSMCVP IGMHCH3 442 DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLP SPLKQTISRPK IGMH CH4 443GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTC VAHEALPNRVTERTVDKST

TABLE 9 Joining Polypeptide for IgA and IgM Class Antibodies Ig Protein Seq. ID No. Sequence Joining 444 QEDERIVLVDNKCKCARITSRIIRS PolypeptideSEDPNEDIVERNIRIIVPLNNRENIS DPTSPLRTRFVYHLSDLCKKC DPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPL VYGGETKMVETALTPDACYPD

CDR and Human Framework Modifications

Riechmann et al., found that the transfer of the CDRs alone (as definedby Kabat (Kabat et al. (supra) and Wu et al., J. Exp. Med., 132,211-250, 1970)) was not sufficient to provide satisfactory antigenbinding activity in the CDR-grafted product. It was found that a numberof framework residues have to be altered so that they correspond tothose of the donor framework region. Proposed criteria for selectingwhich framework residues need to be altered are described inInternational Patent Application WO 90/07861, which is incorporatedherein.

The substitution of non-human CDRs into a human variable domainframework is most likely to result in retention of the CDR's correctspatial orientation if the human variable domain framework adopts thesame or similar conformation to the non-human variable framework fromwhich the CDRs originated. This is achieved by obtaining the humanvariable domains from human antibodies whose framework sequences exhibita high degree of sequence identity with the non-human variable frameworkdomains from which the CDRs were derived. As described above, the heavyand light chain variable framework regions can be derived from the sameor different human antibody sequences. The human antibody sequences canbe the sequences of naturally occurring human antibodies or can beconsensus sequences of several human antibodies. See Kettleborough etal, Protein Engineering 4:773 (1991); Kolbinger et al., ProteinEngineering 6:971 (1993) and Carter et al, WO 92/22653.

Having identified the complementarity determining regions of thenon-human donor immunoglobulin and appropriate human acceptorimmunoglobulins, the next step is to determine which, if any, residuesfrom these components should be substituted to optimize the propertiesof the resulting humanized antibody. In general, substitution of humanamino acid residues with non-human amino acid residues should beminimized, because introduction of non-human residues increases the riskof the antibody eliciting a human-anti-donor-antibody (HADA) response inhumans. Art-recognized methods of determining immune response can beperformed to monitor a HADA response in a particular host or duringclinical trials. Hosts administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HADA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the host using a method known to one in the art,including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 500% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%,sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a donor variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the donor antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g., is within about        3-6 Angstrom of a CDR region as determined by computer        modeling), or    -   (4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly’ interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like. CDR and frameworkregions are as defined by Kabat et al. or Chothia et al., supra. Whenframework residues, as defined by Kabat et al., supra, constitutestructural loop residues as defined by Chothia et al., supra, the aminoacids present in the donor antibody may be selected for substitutioninto the humanized antibody. Residues which are “adjacent to a CDRregion” include amino acid residues in positions immediately adjacent toone or more of the CDRs in the primary sequence of the humanizedimmunoglobulin chain, for example, in positions immediately adjacent toa CDR as defined by Kabat, or a CDR as defined by Chothia (See e.g.,Chothia and Lesk 1 MB 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al, Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to effect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above. In the case of atoms that may form a hydrogen bond, the 3Å is measured between their nuclei, but for atoms that do not form abond, the 3 Å is measured between their Van der Waals surfaces. Hence,in the latter case, the nuclei must be within about 6 Å (3 Å plus thesum of the Van der Waals radii) for the atoms to be considered capableof interacting. In many cases the nuclei will be from 4 or 5 to 6 Åapart. In determining whether an amino acid can interact with the CDRs,it is preferred not to consider the last 8 amino acids of heavy chainCDR 2 as part of the CDRs, because from the viewpoint of structure,these 8 amino acids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

Particular amino acids at several positions in the framework are knownto be capable of interacting with the CDRs in many antibodies (Chothiaand Lesk, supra, Chothia et al., supra and Tramontano et al., J. Mol.Biol. 215:175 (1990), all of which are incorporated herein byreference). Notably, the amino acids at positions 2, 48, 64, and 71 ofthe light chain and 71 and 94 of the heavy chain (numbering according toKabat) are known to be capable of interacting with the CDRs in manyantibodies. The amino acids at positions 35 in the light chain and 93and 103 in the heavy chain are also likely to interact with the CDRs. Atall these numbered positions, choice of the donor amino acid rather thanthe acceptor amino acid (when they differ) to be in the humanizedimmunoglobulin is preferred. On the other hand, certain residues capableof interacting with the CDR region, such as the first 5 amino acids ofthe light chain, may sometimes be chosen from the acceptorimmunoglobulin without loss of affinity in the humanized immunoglobulin.

In one embodiment, a humanized antibody to aP2 is provided comprising atleast one light chain CDR selected from Seq. ID Nos. 7-13, at least oneheavy chain CDR selected from Seq. ID Nos. 14-21, and at least onesubstitution within a human acceptor framework, wherein the substitutionis derived from a donor residue.

In one example a humanized antibody is provided, wherein at least theresidues at one of positions 23, 67, 71, 72, 73, 74, 76, 77, 78, 79, 88,89, 91, 93 and 94 of the variable domain of the heavy chain (Kabatnumbering) are donor residues. In one embodiment, at least the residuesat one of positions 23, 67, 71, 72, 73, 74, 77, 78, 79, 89, and 91 ofthe variable domain of the heavy chain (Kabat numbering) are donorresidues. In one embodiment, at least the residues at one of positions23, 67, 71, 72, 73, 74, 77, 78, 79, 88, 89, 91, 93, and 94 of thevariable domain of the heavy chain (Kabat numbering) are donor residues,but optionally (and in any permutation) one or more of the residues atpositions 23, 67, 71, 72, 73, 74, 77, 78, 79, 88, 89, 91, 93, and 94 mayuse the human acceptor sequence. See for example the sequence given inSeq. ID Nos. 455, 457, 459, 461, and 463.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 1 of theheavy chain variable domain is replaced with an alternative amino acid,for example glutamic acid.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 23 of theheavy chain variable domain is replaced with an alternative amino acid,for example threonine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 23 of theheavy chain is alanine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 67 of theheavy chain variable domain is replaced with an alternative amino acid,for example phenylalanine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 67 of theheavy chain variable domain is valine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 71 of theheavy chain variable domain is replaced with an alternative amino acid,for example lysine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 71 of theheavy chain variable domain is valine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID. No 19 or Seq. ID No. 20, and residue 72 of theheavy chain variable domain is replaced with an alternative amino acid,for example alanine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID. No 19 or Seq. ID No. 20, and residue 72 of theheavy chain variable domain is aspartic acid.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 73 of theheavy chain variable domain is replaced with an alternative amino acid,for example serine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 73 of theheavy chain variable domain is lysine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 74 of theheavy chain variable domain is replaced with an alternative amino acid,for example threonine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 74 of theheavy chain variable domain is serine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 77 of theheavy chain variable domain is replaced with an alternative amino acid,for example threonine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 77 of theheavy chain variable domain is glutamine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 78 of theheavy chain variable domain is replaced with an alternative amino acid,for example valine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 78 of theheavy chain variable domain is phenylalanine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 79 of theheavy chain variable domain is replaced with an alternative amino acid,for example aspartic acid.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 79 of theheavy chain variable domain is serine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 89 of theheavy chain variable domain is replaced with an alternative amino acid,for example threonine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 89 of theheavy chain variable domain is valine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 91 of theheavy chain variable domain is replaced with an alternative amino acid,for example phenylalanine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 91 of theheavy chain variable domain is tyrosine.

In one embodiment, the CDRs are Seq. ID No. 14, Seq. ID No. 16 or Seq.ID No. 17, and Seq. ID No. 19 or Seq. ID No. 20, and residue 23 isalanine, residue 67 is valine, residue 71 is valine, residue 72 isaspartic acid, reside 73 is lysine, reside 74 is serine, residue 77 isglutamine, residue 78 is phenylalanine, residue 79 is serine, residue 89is valine, and residue 91 is tyrosine.

Accordingly, in one example there is provided a humanized antibody,wherein at least the residues at one of positions 2, 3, 36, 37, 58, 63,or 70 of the variable domain of the light chain (Kabat numbering) aredonor residues. In one embodiment, at least the residues at one ofpositions 2, 3, 63, or 70 of the variable domain of the light chain(Kabat numbering) are donor residues. See for example the sequence givenin Seq. ID Nos. 446, 448, 450, and 452.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue2 of the light chain variable domain is replaced with an alternativeamino acid, for example valine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue2 of the light chain variable domain is isoleucine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue3 of the light chain variable domain is replaced with an alternativeamino acid, for example valine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue3 of the light chain variable domain is glutamine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue63 of the light chain variable domain is replaced with an alternativeamino acid, for example lysine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue63 of the light chain variable domain is serine.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue70 of the light chain variable domain is replaced with an alternativeamino acid, for example aspartic acid.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue70 of the light chain variable domain is glutamic acid.

In one embodiment, the CDRs are Seq. ID No. 7, Seq. ID No. 8, and Seq.ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. ID No. 12, and residue2 is isoleucine, residue 3 is glutamine, residue 63 is serine, residue70 is glutamic acid.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative or similarsubstitutions is intended combinations such as, for example, leucinebeing substituted for isoleucine or valine. Other amino acids, which canoften be substituted for one another, include but are not limited to:

phenylalanine, tyrosine and tryptophan (amino acids having aromatic sidechains);

lysine, arginine and histidine (amino acids having basic side chains);

aspartate and glutamate (amino acids having acidic side chains);

asparagine and glutamine (amino acids having amide side chains); and,

cysteine and methionine (amino acids having sulphur-containing sidechains).

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criterion help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common,” as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al, supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the CDRs of Seq.ID Nos. 7-21 as described herein and/or the contact definitionsdescribed herein.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for donor antibodies at that position. For donorantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the donorsequence, which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding could be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence similarity or identity to a human variable frameworkregion sequence or consensus of such sequences. In another embodiment,the variable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98%, or 99%,sequence similarity or identity to a human variable framework regionsequence or consensus of such sequences. In general, however, suchsubstitutions are undesirable.

As used herein, degrees of identity and similarity can be readilycalculated, for example as described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing.Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987,Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991, the BLAST™ software available from NCBI(Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. &States, D. J. 1993, Nature Genet. 3:266-272. Madden, T. L. et al., 1996,Meth. Enzymol. 266:131-141; Altschul, S. F. et al., 1997, Nucleic AcidsRes. 25:3389-3402; Zhang, J. & Madden, T. L. 1997, Genome Res.7:649-656, which are incorporated by reference herein.

A number of reviews discussing CDR-grafted antibodies have beenpublished, including Vaughan et al. (Nature Biotechnology, 16, 535-539,1998), which is incorporated by reference herein.

The anti-aP2 antibodies of the present invention may include furtheradditional binding domains for example as per the molecule DVD-Ig asdisclosed in WO 2007/024715, or the so-called (FabFv)2Fc described inWO2011/030107. Thus antibody as employed herein includes bi, tri ortetra-valent full length antibodies.

Antigen Binding Agents

Antigen binding agents include single chain antibodies (i.e. a fulllength heavy chain and light chain); Fab, modified Fab, Fab′, modifiedFab′, F(ab′)2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VHor VL or VHH) for example as described in WO 2001090190, scFv, bi, trior tetra-valent antibodies. Bis-scFv, diabodies, tribodies, triabodies,tetrabodies and epitope-antigen binding agents of any of the above (seefor example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136;Adair and Lawson, 2005, Drug Design Reviews—Online 2(3), 209-217). Themethods for creating and manufacturing these antibody fragments are wellknown in the art (see for example Verma et al., 1998, Journal ofImmunological Methods, 216, 165-181). The Fab-Fv format was firstdisclosed in WO2009/040562 and the disulphide stabilised versionsthereof, the Fab-dsFv was first disclosed in WO2010/035012. Otherantibody fragments for use in the present invention include the Fab andFab′ fragments described in International patent applicationsWO2005/003169, WO2005/003170, and WO2005/003171. Multi-valent antibodiesmay comprise multiple specificities e.g. bispecific or may bemonospecific (see for example WO 92/22583 and WO05/113605). One suchexample of the latter is a Tri-Fab (or TFM) as described in WO92/22583.

A typical Fab′ molecule comprises a heavy and a light chain pair inwhich the heavy chain comprises a variable region VH, a constant domainCH1 and a natural or modified hinge region and the light chain comprisesa variable region VL and a constant domain CL.

In one embodiment, there is provided a dimer of a Fab′ according to thepresent disclosure to create a F(ab′)2 for example dimerization may bethrough a natural hinge sequence described herein, or derivativethereof, or a synthetic hinge sequence.

An antibody binding domain will generally comprise 6 CDRs, three from aheavy chain and three from a light chain. In one embodiment, the CDRsare in a framework and together form a variable region. Thus in oneembodiment, the antigen binding agent includes a binding domain specificfor aP2 comprising a light chain variable region and a heavy chainvariable region.

It will be appreciated that one or more (for example 1, 2, 3 or 4) aminoacid substitutions, additions and/or deletions may be made to the CDRsor other sequences (e.g. variable domains) provided by the presentinvention, as described above or below, without significantly alteringthe ability of the antibody to bind to aP2. The effect of any amino acidsubstitutions, additions and/or deletions can be readily tested by oneskilled in the art, for example by using the methods described herein,in particular in the Examples.

In one embodiment, one or more (for example 1, 2, 3 or 4) amino acidsubstitutions, additions and/or deletions may be made to the CDRs orframework region employed in the antibody or fragment provided by thepresent invention so that the binding affinity to aP2 is retained,increased, or decreased to an affinity of about ≧10⁻⁷ M. In oneembodiment, provided is a modified humanized antibody whereinmodifications have been made to either the CDRs, framework regions, orboth, in order to decrease the binding affinity, for example, to about≧10⁻⁷ M.

Rabbit Donor CDR/Human Acceptor Framework Anti-aP2 Monoclonal Antibodies

In one aspect of the present invention a humanized anti-aP2 monoclonalantibody derived from an anti-aP2 rabbit CDR/mouse framework hybriddonor monoclonal antibody is provided, wherein the CDRs from theanti-human aP2 protein monoclonal antibody are grafted into human lightchain and heavy chain framework regions.

In one embodiment, the variable light chain Ab 909 VL (Seq. ID No. 445(Table 10, below and FIG. 27)) is the donor light chain sequence usedfor subsequent grafting into a human framework, wherein subsequent CDRand framework modifications may be optionally performed. In oneembodiment, the CDRs provided for in Seq. ID Nos. 7, 8, and 9 derivedfrom the variable light chain provided in Seq. ID No. 445, are graftedinto the human immunoglobulin kappa light chain variable domain of humanimmunoglobulin IGKVA30-JK4, resulting in a humanized light chainvariable region comprising A30 FR1 (Seq. ID No. 40)-CDRL1 (Seq. ID. No.7)-A30 FR2 (Seq. ID No. 41)-CDRL2 (Seq. ID No. 8)-A30 FR3 (Seq. ID No.41)-CDRL3 (Seq. ID. No. 9)-JK4 (Seq. ID No. 148).

In one embodiment, the CDRs provided for in Seq. ID Nos. 7, 8, and 10derived from the variable light chain provided in Seq. ID No. 445, aregrafted into the human immunoglobulin kappa light chain variable domainof human immunoglobulin IGKVA30-JK4, resulting in a humanized lightchain variable region comprising A30 FR1 (Seq. ID No. 40)-CDRL1 (Seq.ID. No. 7)-A30 FR2 (Seq. ID No. 41)-CDRL2 (Seq. ID No. 8)-A30 FR3 (Seq.ID No. 41)-CDRL3 (Seq. ID. No. 10)-JK4 (Seq. ID No. 148), resulting inAb 909 gL13 (Seq. ID No. 487).

In one embodiment, provided is the humanized kappa light chain variableregion 909 gL1 (Seq. ID No. 446 (Table 10, below and FIG. 27)) whereinlight chain variable region donor residues 2V, 3V, 63K, and 70D from Ab909 VL (Seq. ID No. 445) are used as amino acid substitutes for theIGKV30-JK4 amino acids 2I, 3Q, 63S, 70E, resulting in the humanizedkappa light chain 909 gL1 (Seq. ID No. 446 (Table 10, below and FIG.27)).

In one embodiment, provided is the humanized kappa light chain 909 gL1VL+CL (Seq. ID No. 447 (Table 10, below)), wherein the humanized kappalight chain variable region 909 gL1 (Seq. ID No. 446 (Table 10, belowand FIG. 27)) further comprises a human kappa light chain constantregion.

In one embodiment, provided is the humanized kappa light chain 909 gL10(Seq. ID No. 448 (Table 10, below and FIG. 27)) wherein the light chainvariable region donor residues 2V, 3V, 63K, and 70D from Ab 909 VL (Seq.ID No. 445) are used as amino acid substitutes for the IGKV30-JK4 aminoacids 2I, 3Q, 63S, 70E, and a substitution of an alanine in place ofcysteine in the second position of CDRL3 (CDRL3 variant 1 (Seq. ID No.10)) is provided, resulting in the humanized kappa light chain 909 gL10(Seq. ID No. 448 (Table 10, below and FIG. 27)). Alternatively, afurther substitution comprising C88A in FR3 is provided, resulting in Ab909 gL50 (Seq. ID No. 488).

In one embodiment, provided is the humanized kappa light chain 909 gL10VL+CL (Seq. ID No. 449 (Table 10, below)), wherein the humanized kappalight chain variable region 909 gL10 (Seq. ID No. 448 (Table 10, belowand FIG. 27)) further comprises a human kappa light chain constantregion. Alternatively, a further substitution comprising C88A in FR3 isprovided, resulting in Ab909 gL50VL+CL (Seq. ID. No. 490).

In one embodiment, provided is the humanized kappa light chain 909 gL54(Seq. ID No. 450 (Table 10, below)) wherein the light chain variableregion donor residues 2V, 3V, 63K, and 70D from Ab 909 VL (Seq. ID No.445) are used as amino acid substitutes for the IGKV30-JK4 amino acids2I, 3Q, 63S, 70E, and a substitution of an glutamine in place ofcysteine in the second position of CDRL3 (CDRL3 variant 2 (Seq. ID No.11)) is provided, resulting in the humanized kappa light chain 909 gL54(Seq. ID No. 450 (Table 10, below)).

In one embodiment, provided is the humanized kappa light chain 909 gL54VL+CL (Seq. ID No. 451 (Table 10, below)), wherein the humanized kappalight chain variable region 909 gL54 (Seq. ID No. 450 (Table 10, below))further comprises a human kappa light chain constant region.

In one embodiment, provided is the humanized kappa light chain 909 gL55(Seq. ID No. 452 (Table 10, below)) wherein the light chain variableregion donor residues 2V, 3V, 63K, and 70D from Ab 909 VL (Seq. ID No.445) are used as amino acid substitutes for the IGKV30-JK4 amino acids2I, 3Q, 63S, 70E, and a substitution of an histidine in place ofcysteine in the second position of CDRL3 (CDRL3 variant 2 (Seq. ID No.12)) is provided, resulting in the humanized kappa light chain 909 gL55(Seq. ID No. 452 (Table 10, below)).

In one embodiment, provided is the humanized kappa light chain 909 gL55VL+CL (Seq. ID No. 453 (Table 10, below)), wherein the humanized kappalight chain variable region 909 gL55 (Seq. ID No. 452 (Table 10, below))further comprises a human kappa light chain constant region.

TABLE 10 Sequences of Humanized aP2 Light Chain Regions Seq. ID ProteinNo. Sequence Rabbit Ab 909 VL- 445DVVMTQTPASVSEPVGGTVTIKCQASEDTSRYLVWYQQKPGQPPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQCT YGTYAGSFFYSFGGGTEVVVE 909gL1 VL-region 446 DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPKRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQCTY GTYAGSFFYSFGGGTKVEIK 909gL1 VL + CL- 447 DVVMTQSPSSLSASVGDRVTITCQASEDTSRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQCTYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 909 gL10 VL-region 448DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYOQKPGKAPKRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQATY GTYAGSFFYSFGGGTKVEIK 909gL10 VL + CL- 449 DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQATYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 909 gL54 VL-region 450DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPKRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQQTY GTYAGSFFYSFGGGTKVEIK 909gL54 VL + CL- 451 DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQQTYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 909 gL55 VL-region 452DVVMTQSPSSLSASVGDRVTITCQASEDTSRYLVWYQQKPGKAPKRLIYKASTLASGVPSRFKGSGSGTDFTLTTSSLQPEDFATYYCQHTY GTYAGSFFYSFGGGTKVEIK 909gL55 VL + CL- 453 DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQHTYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 909 gL13 VL-region 487DIQMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPKRLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQATY GTYAGSFFYSFGGGTKVEIK 909gL13 VL + CL- 489 DIQMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQATYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 909 gL50 VL-region 488DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPKRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYAQATY GTYAGSFFYSFGGGTKVEIK 909gL50 VL + CL- 490 DVVMTQSPSSLSASVGDRVTITCQASEDISRYLVWYQQKPGKAPK regionRLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYAQATYGTYAGSFFYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

For example, the variable heavy chain Ab 909 VH (Seq. ID No. 454 (Table11, below and FIG. 28)) is the donor heavy chain sequence used forsubsequent grafting into a human framework, wherein subsequent CDR andframework modifications may be optionally performed. In one embodiment,the variable heavy CDRs provided for in Seq. ID. Nos. 14, 16, and 19derived from the variable heavy chain provide in Seq. ID No. 454, aregrafted into the human immunoglobulin heavy chain variable domain ofhuman immunoglobulin IGHV4-04-JH4 (Seq. ID No. 481 (FIG. 28)), resultingin a humanized heavy chain variable region comprising 4-04 FR1 (Seq. IDNo. 357)-CDRH1 (Seq. ID No. 14)-4-04 FR2 (Seq. ID No. 358)-CDRH2 (Seq.ID No. 16)-4-04 FR3 (Seq. ID No. 359)-CDRH3 (Seq. ID No. 19)-JH4 (Seq.ID No. 405).

In one embodiment, the humanized heavy chain variable region 909 gH1variable region is provided (Seq. ID No. 455 (Table 11, below and FIG.28)) wherein the heavy chain variable region donor residues 23T, 67F,71K, 72A, 73S, 74T, 77T, 78V, 79D, 89T, 91F from Ab 909 VH (Seq. ID No.454) are used as amino acid substitutes for the IGHV4-04-JK4 amino acids23A, 67V, 71V, 72D, 73K, 74S, 77Q, 78F, 79S, 89V, 91Y, and 1E issubstituted for the IGHV4-04-JK4 amino acid 1Q, and a two amino acidresidue amino acid gap in framework 3, in the loop between beta sheetstrands D and E at amino acids 75 and 76 is maintained, resulting in thehumanized heavy chain 909 gH1 (Seq. ID No. 455 (Table 11, below and FIG.28)).

In one embodiment, the humanized IgG4 heavy chain 909 gH1 VH+IgG4PConstant is provided (Seq. ID No. 456 (Table 11, below)), wherein thehumanized heavy chain variable region 909 gH1 (Seq. ID No. 455 (Table11, below and FIG. 28)) further comprises a human IgG4P constant region.IgG4P as employed herein is a mutation of the wild-type IgG4 isotypewhere amino acid 241 is replaced by proline see for example where serineat position 241 has been changed to proline as described in Angal etal., Molecular Immunology, 1993, 30 (1), 105-108.

In one embodiment, the humanized heavy chain variable region 909 gH14variable region is provided (Seq. ID No. 457 (Table 11, below and FIG.28)) wherein the heavy chain variable region donor residues 67F, 71K,72A, 73S, 74T, 77T, 78V, 79D, 89T, 91F from Ab 909 VH (Seq. ID No. 454)are used as amino acid substitutes for the IGHV4-04-JK4 amino acids 67V,71V, 72D, 73K, 74S, 77Q, 78F, 79S, 89V, 91Y, and 1E is substituted forthe IGHV4-04-JK4 amino acid 1Q, resulting in the humanized heavy chain909 gH4 (Seq. ID No. 457 (Table 11, below and FIG. 28)).

In one embodiment, the humanized IgG4 heavy chain 909 gH14 VH+IgG4PConstant is provided (Seq. ID No. 458 (Table 11, below)), wherein thehumanized heavy chain variable region 909 gH14 (Seq. ID No. 457 (Table11, below and FIG. 28)) further comprises a human IgG4P constant region.IgG4P as employed herein is a mutation of the wild-type IgG4 isotypewhere amino acid 241 is replaced by proline see for example where serineat position 241 has been changed to proline as described in Angal etal., Molecular Immunology, 1993, 30 (1), 105-108.

In one embodiment, the humanized heavy chain variable region 909 gH15variable region (Seq. ID No. 459 (Table 11, below and FIG. 28)) isprovided wherein the heavy chain variable region donor residues 23T,67F, 71K, 72A, 73S, 74T, 77T, 78V, 79D, 89T, 91F from Rabbit Ab 909 VH(Seq. ID No. 454) are used as amino acid substitutes for theIGHV4-04-JK4 amino acids 23A, 67V, 71V, 72D, 73K, 74S, 77Q, 78F, 79S,89V, 91Y, and 1E is substituted for the IGHV4-04-JK4 amino acid 1Q, andthere is a substitution of a serine in place of cysteine in the tenthposition of CDRH2 (CDRH2 variant 1 (Seq. ID No. 17)) and a substitutionof a glutamic acid in place of aspartic acid in the fourth position ofCDRH3 (CDRH3 variant 1 (Seq. ID No. 20), resulting in the humanizedheavy chain 909 gH15 VH region (Seq. ID No. 459 (Table 11, below andFIG. 28)).

In one embodiment, the humanized IgG4 heavy chain 909 gH15 VH+IgG4PConstant (Seq. ID No. 460 (Table 11, below)) is provided, wherein thehumanized heavy chain variable region 909 gH15 VH (Seq. ID No. 459(Table 11, below and FIG. 28)) further comprises a human IgG4P constantregion. IgG4P as employed herein is a mutation of the wild-type IgG4isotype where amino acid 241 is replaced by proline see for examplewhere serine at position 241 has been changed to proline as described inAngal et al., Molecular Immunology, 1993, 30 (1), 105-108.

In one embodiment, the humanized heavy chain variable region 909 gH61variable region (Seq. ID No. 461 (Table 11, below and FIG. 28)) isprovided wherein the heavy chain variable region donor residues 71K,73S, 78V from Rabbit Ab 909 VH (Seq. ID No. 454) are used as amino acidsubstitutes for the IGHV4-04-JK4 amino acids 71V, 73K, 78F, and 1E issubstituted for the IGHV4-04-JK4 amino acid 1Q, resulting in thehumanized heavy chain 909 gH61 VH region (Seq. ID No. 461 (Table 11,below and FIG. 28)).

In one embodiment, the humanized IgG4 heavy chain 909 gH61 VH+IgG4PConstant (Seq. ID No. 462 (Table 11, below)) is provided, wherein thehumanized heavy chain variable region 909 gH61 VH (Seq. ID No. 461(Table 11, below and FIG. 28)) further comprises a human IgG4P constantregion. IgG4P as employed herein is a mutation of the wild-type IgG4isotype where amino acid 241 is replaced by proline see for examplewhere serine at position 241 has been changed to proline as described inAngal et al., Molecular Immunology, 1993, 30 (1), 105-108.

In one embodiment, the humanized heavy chain variable region 909 gH62variable region (Seq. ID No. 463 (Table 11, below and FIG. 28)) isprovided wherein the heavy chain variable region donor residues 71K,73S, 78V from Rabbit Ab 909 VH (Seq. ID No. 454) are used as amino acidsubstitutes for the IGHV4-04-JK4 amino acids 71V, 73K, 78F, and 1E issubstituted for the IGHV4-04-JK4 amino acid 1Q, and there is asubstitution of a serine in place of cysteine in the tenth position ofCDRH2 (CDRH2 variant 1 (Seq. ID No. 17)) and a substitution of aglutamic acid in place of aspartic acid in the fourth position of CDRH3(CDRH3 variant 1 (Seq. ID No. 20), resulting in the humanized heavychain 909 gH62 VH region (Seq. ID No. 463 (Table 11, below and FIG.28)).

In one embodiment, the humanized IgG4 heavy chain 909 gH62 VH+IgG4PConstant (Seq. ID No. 464 (Table 11, below)) is provided, wherein thehumanized heavy chain variable region 909 gH62 VH (Seq. ID No. 463(Table 11, below and FIG. 28)) further comprises a human IgG4P constantregion. IgG4P as employed herein is a mutation of the wild-type IgG4isotype where amino acid 241 is replaced by proline see for examplewhere serine at position 241 has been changed to proline as described inAngal et al., Molecular Immunology, 1993, 30 (1), 105-108.

TABLE 11 Sequences of Humanized aP2 Heavy Chain Regions Seq. ID ProteinNo. Sequence Rabbit Ab 909 VH 454QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYYMSWVRQAPGKGLE regionWIGHYPSGSTYCASWAKGRFTISKASTTVDLKITSPTTEDTATYFCARPDNDGTSGYLSGFGLWGQGTLVTVSS 909gH1 VH region 455EVQLQESGPGLVKPSGTLSLTCTVSGFSLSTYYMSWVRQPPGKGLEWIGIIYPSGSTYCASWAKGRFTISKASTTVDLKLSSVTAADTATYFCARPDNDGTSGYLSGFGLWGQGTLVTVSS 909gH1 IgG4 VH + 456EVQLQESGPGLVKPSGTLSLTCTVSGFSLSTYYMSWVRQPPGKGL human γ-4P constantEWIGIIYPSGSTYCASWAKGRFTISKASTTVDLKLSSVTAADTATYFCARPDNDGTSGYLSGFGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK 909gH14 VH region 457EVQLQESGPG LVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGLEWIGIIYPSGSTYCASWAKGRFTISKASTKNTVDLKLSSVTAADTATYFCARPDNDGTSGYLSGFGLWGQGTLVTVSS 909gH14 IgG4 VH + 458EVQLQESGPGLVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGL human γ-4P constantEWIGIIYPSGSTYCASWAKGRFTISKASTKNTVDLKLSSVTAADTATYFCARPDNDGTSGYLSGFGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 909 gH15 VH region 459EVQLQESGPGLVKPSGTLSLTCTVSGFSLSTYYMSWVRQPPGKGLEWIGIIYPSGSTYSASWAKGRFTISKASTKNTVDLKLSSVTAADTATYFCARPDNEGTSGYLSGFGLWGQGTLVTVSS 909gH15 IgG4 VH + 460EVQLQESGPGLVKPSGTLSLTCTVSGFSLSTYYMSWVRQPPGKGL human γ-4P constantEWIGIIYPSGSTYSASWAKGRFTISKASTKNTVDLKLSSVTAADTATYFCARPDNEGTSGYLSGFGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 909 gH61 VH region 461EVQLQESGPGLVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGLEWIGIIYPSGSTYCASWAKGRVTISKDSSKNQVSLKLSSVTAADTAVYYCARPDNDGTSGYLSGFGLWGQGTLVTVSS 909gH61 IgG4 VH + 462EVQLQESGPGLVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGL human γ-4P constantEWIGIIYPSGSTYCASWAKGRVTISKDSSKNQVSLKLSSVTAADTAVYYCARPDNDGTSGYLSGFGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 909 gH62 VH region 463EVQLQESGPGLVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGLEWIGIIYPSGSTYSASWAKGRVTISKDSSKNQVSLKLSSVTAADTAVYYCARPDNEGTSGYLSGFGLWGQGTLVTVSS 909gH62 IgG4 VH + 464EVQLQESGPGLVKPSGTLSLTCAVSGFSLSTYYMSWVRQPPGKGL human γ-4P constantEWIGIIYPSGSTYSASWAKGRVTISKDSSKNQVSLKLSSVTAADTAVYYCARPDNEGTSGYLSGFGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In one embodiment the disclosure provides an antibody sequence which is80% similar or identical to a sequence disclosed herein, for example85%, 900/%, 91%, 92%, 93/%, 94%, 95% 96%, 97%, 98% or 99% over part orwhole of the relevant sequence, for example a variable domain sequence,a CDR sequence or a variable domain sequence excluding the CDRs. In oneembodiment the relevant sequence is selected from Seq. ID Nos. 446, 447,448, 487, 488, 489, 490, 449, 450, 451, 452, 453, 455, 456, 457, 458,459, 460, 461, 462, 463, or 464. In one embodiment the disclosureprovides an antibody sequence which has one or more (for example, 1, 2,3, or 4) amino acid substitutions, additions, or deletions in therelevant sequence, for example a variable domain sequence, a CDRsequence or a variable domain sequence excluding the CDRs selected fromSeq. ID Nos. 446, 447, 448, 487, 488, 489, 490, 449, 450, 451, 452, 453,455, 456, 457, 458, 459, 460, 461, 462, 463, or 464.

In one embodiment, the present invention provides an antibody moleculewhich binds human aP2 comprising a light chain, wherein the variabledomain of the light chain comprises a sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identity or similarityto a sequence selected from Seq. ID Nos. 446, 448, 487, 488, 450, or452. In one embodiment, the present invention provides an antibodymolecule which binds human aP2 comprising a light chain, wherein thevariable domain of the light chain comprises a sequence having one ormore (for example, 1, 2, 3, or 4) amino acid substitutions, additions,or deletions in its sequence compared to Seq. ID Nos. 446, 448, 487,488, 450, or 452.

In one embodiment the present invention provides an antibody moleculewhich binds human aP2 wherein the antibody has a heavy chain variabledomain which is at least 80% 90/0, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%or 99% similar or identical to a sequence selected from Seq. ID Nos.455, 457, 459, 461 or 463. In one embodiment the present inventionprovides an antibody molecule which binds human aP2 wherein the antibodyhas a heavy chain variable domain which has one or more (for example, 1,2, 3, or 4) amino acid substitutions, additions, or deletions in itssequence compared to Seq. ID Nos. 455, 467, 459, 461 or 463.

A suitable framework region for the light chain of the humanizedantibody of the present invention is derived from the human germlinesub-group IgKV1-17 (A30) (Seq. ID Nos. 40-42) together with JK4 (Seq. IDNo. 148). Accordingly, in one example there is provided a humanizedantibody comprising the sequence given in Seq. ID No. 7 for CDR-L1, thesequence given in Seq. ID No. 8 for CDR-L2 and the sequence selectedfrom Seq. ID Nos. 9, 10, 11, or 12 for CDRL3, wherein the light chainframework region is derived from the human subgroup IGKV1-17 (A30) (Seq.ID Nos. 40-42) together with JK4 (Seq. ID No. 148). The JK4 sequence isas follows: FGGGTKVEIK (Seq. ID No. 148). In one example the light chainvariable domain of the antibody comprises the sequence selected fromSeq. ID Nos. 446, 448, 487, 488, 450, and 452.

A suitable framework region for the heavy chain variable region of thehumanized antibody of the present invention is derived from the humangermline sub-group IGHV 4-04 (Seq. ID Nos. 357, 358, and 359) togetherwith JH4 (Seq. ID No. 405). Accordingly, in one example there isprovided a humanized antibody comprising the sequence given in Seq. IDNo. 14 for CDR-H1, a sequence selected from Seq. ID. Nos. 16 or 17 forCDR-H2 and the sequence selected from Seq. ID Nos. 19 or 20 for CDR-H3,wherein the heavy chain framework region is derived from the humansubgroup IGHV 4-04 (Seq. ID Nos. 357, 358, and 359) together with JH4(Seq. ID No. 405). The JH4 sequence is as follows: WGQGTLVTVSS (Seq. IDNo. 405).

In one embodiment the antibody molecule of the present disclosure is aFab, Fab′, or F(ab′)2 antibody fragment comprising a light chainvariable region selected from Seq. ID Nos. 446, 448, 487, 488, 450, or452, and a heavy chain variable region selected from Seq. ID Nos. 455,457, 459, 461, or 463. In one embodiment the antibody molecule of thepresent disclosure is a Fab, Fab′, or F(ab′)2 antibody fragmentcomprising a light chain variable region given in Seq. ID No. 446, and aheavy chain variable region given in Seq. ID No. 455. In one embodimentthe antibody molecule of the present disclosure is a Fab, Fab′, orF(ab′)2 antibody fragment comprising a light chain variable region givenin Seq. ID No. 448, and a heavy chain variable region given in Seq. IDNo. 455. In one embodiment the antibody molecule of the presentdisclosure is a Fab, Fab′, or F(ab′)2 antibody fragment comprising alight chain variable region given in Seq. ID No. 450, and a heavy chainvariable region given in Seq. ID No. 455. In one embodiment the antibodymolecule of the present disclosure is a Fab, Fab′, or F(ab′)2 antibodyfragment comprising a light chain variable region given in Seq. ID No.452, and a heavy chain variable region given in Seq. ID No. 455. In oneembodiment the antibody molecule of the present disclosure is a Fab,Fab′, or F(ab′)2 antibody fragment comprising a light chain variableregion given in Seq. ID No. 487, and a heavy chain variable region givenin Seq. ID No. 455. In one embodiment the antibody molecule of thepresent disclosure is a Fab, Fab′, or F(ab′)2 antibody fragmentcomprising a light chain variable region given in Seq. ID No. 488, and aheavy chain variable region given in Seq. ID No. 455. In one embodiment,the antibody molecule of the present disclosure is a Fab, Fab′, orF(ab′)2 antibody fragment comprising a light chain variable region givenin Seq. ID No. 446 and a heavy chain variable region given in Seq. IDNo. 459. In one embodiment the antibody molecule of the presentdisclosure is a Fab, Fab′, or F(ab′)2 antibody fragment comprising alight chain variable region given in Seq. ID No. 448, and a heavy chainvariable region given in Seq. ID No. 459. In one embodiment, theantibody molecule of the present disclosure is a Fab, Fab′, or F(ab′)2antibody fragment comprising a light chain variable region given in Seq.ID No. 450 and a heavy chain variable region given in Seq. ID No. 459.In one embodiment, the antibody molecule of the present disclosure is aFab, Fab′, or F(ab′)2 antibody fragment comprising a light chainvariable region given in Seq. ID No. 452 and a heavy chain variableregion given in Seq. ID No. 459. In one embodiment, the antibodymolecule of the present disclosure is a Fab, Fab′, or F(ab′)2 antibodyfragment comprising a light chain variable region given in Seq. ID No.487 and a heavy chain variable region given in Seq. ID No. 459. In oneembodiment, the antibody molecule of the present disclosure is a Fab,Fab′, or F(ab′)2 antibody fragment comprising a light chain variableregion given in Seq. ID No. 488 and a heavy chain variable region givenin Seq. ID No. 459. In one embodiment, the antibody molecule of thepresent disclosure is a Fab, Fab′, or F(ab′)2 antibody fragmentcomprising a light chain variable region given in Seq. ID No. 446 and aheavy chain variable region given in Seq. ID No. 457. In one embodimentthe antibody molecule of the present disclosure is a Fab, Fab′, orF(ab′)2 antibody fragment comprising a light chain variable region givenin Seq. ID No. 448, and a heavy chain variable region given in Seq. IDNo. 457. In one embodiment, the antibody molecule of the presentdisclosure is a Fab, Fab′, or F(ab′)2 antibody fragment comprising alight chain variable region given in Seq. ID No. 450 and a heavy chainvariable region given in Seq. ID No. 457. In one embodiment, theantibody molecule of the present disclosure is a Fab, Fab′, or F(ab′)2antibody fragment comprising a light chain variable region given in Seq.ID No. 452 and a heavy chain variable region given in Seq. ID No. 457.In one embodiment, the antibody molecule of the present disclosure is aFab, Fab′, or F(ab′)2 antibody fragment comprising a light chainvariable region given in Seq. ID No. 487 and a heavy chain variableregion given in Seq. ID No. 457. In one embodiment, the antibodymolecule of the present disclosure is a Fab, Fab′, or F(ab′)2 antibodyfragment comprising a light chain variable region given in Seq. ID No.488 and a heavy chain variable region given in Seq. ID No. 457. In oneembodiment, the antibody molecule of the present disclosure is a Fab,Fab′, or F(ab′)2 antibody fragment comprising a light chain variableregion given in Seq. ID No. 446 and a heavy chain variable region givenin Seq. ID No. 461. In one embodiment the antibody molecule of thepresent disclosure is a Fab, Fab′, or F(ab′)2 antibody fragmentcomprising a light chain variable region given in Seq. ID No. 448, and aheavy chain variable region given in Seq. ID No. 461. In one embodiment,the antibody molecule of the present disclosure is a Fab, Fab′, orF(ab′)2 antibody fragment comprising a light chain variable region givenin Seq. ID No. 450 and a heavy chain variable region given in Seq. IDNo. 461. In one embodiment, the antibody molecule of the presentdisclosure is a Fab, Fab′, or F(ab′)2 antibody fragment comprising alight chain variable region given in Seq. ID No. 452 and a heavy chainvariable region given in Seq. ID No. 461. In one embodiment, theantibody molecule of the present disclosure is a Fab, Fab′, or F(ab′)2antibody fragment comprising a light chain variable region given in Seq.ID No. 487 and a heavy chain variable region given in Seq. ID No. 461.In one embodiment, the antibody molecule of the present disclosure is aFab, Fab′, or F(ab′)2 antibody fragment comprising a light chainvariable region given in Seq. ID No. 488 and a heavy chain variableregion given in Seq. ID No. 461. In one embodiment, the antibodymolecule of the present disclosure is a Fab, Fab′, or F(ab′)2 antibodyfragment comprising a light chain variable region given in Seq. ID No.446 and a heavy chain variable region given in Seq. ID No. 463. In oneembodiment the antibody molecule of the present disclosure is a Fab,Fab′, or F(ab′)2 antibody fragment comprising a light chain variableregion given in Seq. ID No. 448, and a heavy chain variable region givenin Seq. ID No. 463. In one embodiment, the antibody molecule of thepresent disclosure is a Fab, Fab′, or F(ab′)2 antibody fragmentcomprising a light chain variable region given in Seq. ID No. 450 and aheavy chain variable region given in Seq. ID No. 463. In one embodiment,the antibody molecule of the present disclosure is a Fab, Fab′, orF(ab′)2 antibody fragment comprising a light chain variable region givenin Seq. ID No. 452 and a heavy chain variable region given in Seq. IDNo. 463. In one embodiment, the antibody molecule of the presentdisclosure is a Fab, Fab′, or F(ab′)2 antibody fragment comprising alight chain variable region given in Seq. ID No. 487 and a heavy chainvariable region given in Seq. ID No. 463. In one embodiment, theantibody molecule of the present disclosure is a Fab, Fab′, or F(ab′)2antibody fragment comprising a light chain variable region given in Seq.ID No. 488 and a heavy chain variable region given in Seq. ID No. 463.

In one embodiment the antibody molecule of the present disclosure is afull length IgG1 antibody comprising the variable regions shown in Seq.ID Nos. 446, 448, 487, 488, 450, or 452 for the light chain and Seq. IDNos. 455, 457, 459, 461, or 463 for the heavy chain.

In one embodiment the antibody molecule of the present disclosure is afull length IgG4 antibody comprising the variable regions shown in Seq.ID Nos. 446, 448, 487, 488, 450, or 452 for the light chain and Seq. IDNos. 455, 457, 459, 461, or 463 for the heavy chain.

In one embodiment the antibody molecule of the present disclosure is afull length IgG4P antibody comprising the variable regions shown in Seq.ID Nos. 446, 448, 487, 488, 450, or 452 for the light chain and Seq. IDNos. 455, 457, 459, 461, or 463 for the heavy chain. In one embodimentthe antibody molecule has a light chain comprising a sequence selectedfrom Seq. ID. Nos. 447, 449, 489, 490, 451, or 453 for the light chainand a sequence selected from Seq. ID Nos. 456, 458, 460, 462, or 464 forthe heavy chain. In one embodiment the antibody according to the presentdisclosure is provided as aP2 binding antibody fusion protein whichcomprises an immunoglobulin moiety, for example a Fab or Fab′ fragment,and one or two single domain antibodies (dAb) linked directly orindirectly thereto, for example as described in WO2009/040562,WO2010035012, WO2011/030107, WO2011/061492 and WO2011/086091 allincorporated herein by reference.

In one embodiment the fusion protein comprises two domain antibodies,for example as a variable heavy (VH) and variable light (VL) pairing,optionally linked by a disulphide bond.

The antibody fragment of the present invention includes Fab, Fab′,F(ab′)2, scFv, diabody, scFAb, dFv, single domain light chainantibodies, dsFv, a peptide comprising CDR, and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and antigen binding activity, in which about a half of the N-terminalside of H chain and the entire L chain, among fragments obtained bytreating IgG with a protease, papain (cut at an amino acid residue atposition 224 of the H chain), are bound together through a disulfidebond.

The Fab of the present invention can be obtained by treating a humanCDR-grafted antibody of the present invention, which specifically reactswith aP2, with a protease, papain. Also, the Fab can be produced byinserting DNA encoding Fab of the antibody into an expression vector forprokaryote or an expression vector for eukaryote, and introducing thevector into a prokaryote or eukaryote to express the Fab.

An F(ab′)2 is an antibody fragment having a molecular weight of about100,000 and antigen binding activity, which is slightly larger than theFab bound via a disulfide bond of the hinge region, among fragmentsobtained by treating IgG with a protease, pepsin.

The F(ab′)2 of the present invention can be obtained by treating a humanCDR-grafted antibody which specifically reacts with aP2, with aprotease, pepsin. Also, the F(ab′)2 can be produced by binding Fab′described below via a thioether bond or a disulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cutting adisulfide bond of the hinge region of the F(ab′)2.

The Fab′ of the present invention can be obtained by treating theF(ab′)2 which specifically reacts with aP2, with a reducing agent,dithiothreitol. Also, the Fab′ of the present invention can be producedby inserting DNA encoding an Fab′ of a human CDR-grafted antibody of thepresent invention which specifically reacts with aP2 into an expressionvector for prokaryote or an expression vector for eukaryote, andintroducing the vector into a prokaryote or eukaryote to express theFab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (P) of 12 ormore residues and which has an antigen-binding activity.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a human CDR-grafted antibody which specificallyreacts with aP2 of the present invention, constructing DNA encodingscFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment in which scFv's having the same ordifferent antigen binding specificity forms a dimer, and has an divalentantigen binding activity to the same antigen or two specific antigenbinding activities to different antigens.

The diabody of the present invention, for example, a divalent diabodywhich specifically reacts with aP2, can be produced by obtaining cDNAsencoding VH and VL of an antibody which specifically reacts with aP2,constructing DNA encoding scFv having a polypeptide linker of 3 to 10residues, inserting the DNA into an expression vector for prokaryote oran expression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residue,which is substituted with a cysteine residue, can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. (Protein Engineering, 7, 697(1994)).

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a human CDR-grafted antibody which specificallyreacts with aP2 of the present invention, constructing DNA encodingdsFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the dsFv.

A peptide comprising CDR is constituted by including at least one regionof H chain and L chain CDRs. Plural CDRs can be bound directly or via anappropriate peptide linker.

The peptide comprising CDR of the present invention can be produced byobtaining cDNA encoding CDR of VH and VL of a human CDR-grafted antibodywhich specifically reacts with aP2, constructing DNA encoding CDR,inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then by introducing the expressionvector into a prokaryote or eukaryote to express the peptide. Also, thepeptide comprising CDR can also be produced by a chemical synthesismethod such as an Fmoc method (fluorenylmethoxycarbonyl method), a tBocmethod (t-butyloxycarbonyl method), or the like.

The antibody of the present invention includes antibody derivatives inwhich a radioisotope, a protein, an agent or the like is chemically orgenetically conjugated to the antibody of the present invention.

The antibody derivatives of the present invention can be produced bychemically conjugating a radioisotope, a protein or an agent to theN-terminal side or C-terminal side of an H chain or an L chain of anantibody or antibody fragment which specifically reacts with aP2, to anappropriate substituent group or side chain of the antibody or antibodyfragment or to a sugar chain in the antibody or antibody fragment(Antibody Engineering Handbook, edited by Osamu Kanemitsu, published byChijin Shokan (1994)).

Also, it can be genetically produced by linking a DNA encoding theantibody or the antibody fragment of the present invention whichspecifically reacts with aP2 to other DNA encoding a protein to bebound, inserting the DNA into an expression vector, and introducing theexpression vector into a host cell.

The radioisotope includes 131I, 125I and the like, and it can beconjugated to the antibody by, e.g., a chloramine T method.

The agent is preferably a low molecular weight compound. Examplesinclude anticancer agents such as alkylating agents (e.g., nitrogenmustard, cyclophosphamide), metabolic antagonists (e.g., 5-fluorouracil,methotrexate), antibiotics (e.g., daunomycin, bleomycin, mitomycin C,daunorubicin, doxorubicin), plant alkaloids (e.g., vincristine,vinblastine, vindesine), hormone drugs (e.g., tamoxifen, dexamethasone),and the like (Clinical Oncology, edited by Japanese Society of ClinicalOncology, published by Cancer and Chemotherapy (1996));anti-inflammatory agents such as steroid agents (e.g., hydrocortisone,prednisone), non-steroidal drugs (e.g., aspirin, indomethacin),immunomodulators (e.g., aurothiomalate, penicillamine),immunosuppressing agents (e.g., cyclophosphamide, azathioprine) andantihistaminic agents (e.g., chlorpheniramine maleate, clemastine)(Inflammation and Anti-inflammatory Therapy, Ishiyaku Shuppan (1982));and the like. The method for conjugating daunomycin to an antibodyincludes a method in which daunomycin and an amino group of an antibodyare conjugated via glutaraldehyde, a method in which an amino group ofdaunomycin and a carboxyl group of an antibody are conjugated via awater-soluble carbodiimide, and the like.

Also, in order to inhibit cancer cells directly, a toxin such as ricin,diphtheria toxin and the like, can be used. For example, a fusionantibody with a protein can be produced by linking a cDNA encoding anantibody or antibody fragment to other cDNA encoding the protein,constructing DNA encoding the fusion antibody, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing it into a prokaryote or eukaryote to express thefusion antibody.

Further contemplated herein are antibody fragments or antigen bindingagents including fusions of binding agents, for example immunoglobulinlike fragments and agents such as diabodies, scAbs, bispecificfragments, triabodies, Fab-Fv-Fv, Fab-Fv, tribody, (Fab-Fv)2-Fc, andantibody fragments or portions, such as CDRs or antibody loops includingCDRs, which are grafted onto non-Ig frameworks such as fibronectin orleucine zippers, as descried in Binz et al., (2005) Nat. Biotech.23:1257-1268, incorporated in its entirety herein.

Conjugated Anti-aP2 Monoclonal Antibodies and Antigen Binding Agents

If desired, an antibody or antigen binding agent for use in the presentinvention may be conjugated to one or more effector molecule(s). It willbe appreciated that the effector molecule may comprise a single effectormolecule or two or more such molecules so linked as to form a singlemoiety that can be attached to the antibodies of the present invention.Where it is desired to obtain an antibody fragment linked to an effectormolecule, this may be prepared by standard chemical or recombinant DNAprocedures in which the antibody fragment is linked either directly orvia a coupling agent to the effector molecule. Techniques forconjugating such effector molecules to antibodies are well known in theart (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinsonet al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev.,62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83,67-123). Particular chemical procedures include, for example, thosedescribed in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03/031581. Alternatively, where the effector molecule is a protein orpolypeptide the linkage may be achieved using recombinant DNAprocedures, for example as described in WO 86/01533 and EP0392745.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins, biologically active proteins, forexample enzymes, other antibody or antibody fragments, antigen bindingagents, synthetic (including PEG) or naturally occurring polymers,nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof,radionuclides, particularly radioiodide, radioisotopes, chelated metals,nanoparticles and reporter groups such as fluorescent compounds orcompounds which may be detected by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxicagents including any agent that is detrimental to (e.g. kills) cells.Examples include combrestatins, dolastatins, epothilones, staurosporin,maytansinoids, spongistatins, rhizoxin, halichondrins, roridins,hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g. vincristine andvinblastine).

Other effector molecules may include chelated radionuclides such as111In and 90Y, Lu177, Bismuth213, Californium252, Iridium192 andTungsten188/Rhenium188; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymesof interest include, but are not limited to, proteolytic enzymes,hydrolases, lyases, isomerases, transferases. Proteins, polypeptides andpeptides of interest include, but are not limited to, immunoglobulins,toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, a protein such as insulin, tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, or, a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF)or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions, which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude 125I, 131I, 111In and 99Tc.

In another example the effector molecule may increase the half-life ofthe antibody in vivo, and/or reduce immunogenicity of the antibodyand/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO05/117984.

In one embodiment a half-life provided by an effector molecule which isindependent of aP2 or an anti-human aP2 antibody is advantageous.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Specific optional substituents, which may be present on theabove-mentioned synthetic polymers, include one or more hydroxy, methylor methoxy groups.

Specific examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran,glycogen or derivatives thereof.

In one embodiment the polymer is albumin or a fragment thereof, such ashuman serum albumin or a fragment thereof. In one embodiment the polymeris a PEG molecule.

“Derivatives” as used herein in regard to conjugates is intended toinclude reactive derivatives, for example thiol-selective reactivegroups such as maleimides and the like. The reactive group may be linkeddirectly or through a linker segment to the polymer. It will beappreciated that the residue of such a group will in some instances formpart of the product as the linking group between the antibody fragmentand the polymer.

The size of the natural or synthetic polymer may be varied as desired,but will generally be in an average molecular weight range from 500 Dato 50000 Da, for example from 5000 to 40000 Da such as from 20000 to40000 Da. The polymer size may in particular be selected on the basis ofthe intended use of the product for example ability to localize tocertain tissues such as tumors or extend circulating half-life (forreview see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545).Thus, for example, where the product is intended to leave thecirculation and penetrate tissue, for example for use in the treatmentof a tumour, it may be advantageous to use a small molecular weightpolymer, for example with a molecular weight of around 5000 Da. Forapplications where the product remains in the circulation, it may beadvantageous to use a higher molecular weight polymer, for examplehaving a molecular weight in the range from 20000 Da to 40000 Da.

Suitable polymers include a polyalkylene polymer, such as apoly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or aderivative thereof, and especially with a molecular weight in the rangefrom about 15000 Da to about 40000 Da.

In one example antibodies for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods (see for example U.S. Pat. No.5,219,996; U.S. Pat. No. 5,667,425; WO98/25971, WO2008/038024). In oneexample the antibody molecule of the present invention is a modified Fabfragment wherein the modification is the addition to the C-terminal endof its heavy chain one or more amino acids to allow the attachment of aneffector molecule. Suitably, the additional amino acids form a modifiedhinge region containing one or more cysteine residues to which theeffector molecule may be attached. Multiple sites can be used to attachtwo or more PEG molecules.

Suitably PEG molecules are covalently linked through a thiol group of atleast one cysteine residue located in the antibody fragment. Eachpolymer molecule attached to the modified antibody fragment may becovalently linked to the sulphur atom of a cysteine residue located inthe fragment. The covalent linkage will generally be a disulphide bondor, in particular, a sulphur-carbon bond. Where a thiol group is used asthe point of attachment appropriately activated effector molecules, forexample thiol selective derivatives such as maleimides and cysteinederivatives may be used. An activated polymer may be used as thestarting material in the preparation of polymer-modified antibodyfragments as described above.

The activated polymer may be any polymer containing a thiol reactivegroup such as an α-halocarboxylic acid or ester, e.g. iodoacetamide, animide, e.g. maleimide, a vinyl sulphone or a disulphide. Such startingmaterials may be obtained commercially (for example from Nektar,formerly Shearwater Polymers Inc., Huntsville, Ala., USA) or may beprepared from commercially available starting materials usingconventional chemical procedures. Particular PEG molecules include 20Kmethoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; RappPolymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerlyShearwater).

In one embodiment, the antibody is a modified Fab fragment, Fab′fragment or diFab which is PEGylated, i.e. has PEG(poly(ethyleneglycol)) covalently attached thereto, e.g. according tothe method disclosed in EP 0948544 or EP1090037 [see also“Poly(ethyleneglycol) Chemistry, Biotechnical and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York,“Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J.Milton Harris and S. Zalipsky (eds), American Chemical Society,Washington D.C. and “Bioconjugation Protein Coupling Techniques for theBiomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, NewYork; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002,54:531-545]. In one example PEG is attached to a cysteine in the hingeregion. In one example, a PEG modified Fab fragment has a maleimidegroup covalently linked to a single thiol group in a modified hingeregion. A lysine residue may be covalently linked to the maleimide groupand to each of the amine groups on the lysine residue may be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab fragment may therefore be approximately 40,000 Da.

Particular PEG molecules include 2-[3-(N-maleimido)propionamido]ethylamide of N,N′-bis(methoxypoly(ethylene glycol) MW 20,000) modifiedlysine, also known as PEG2MAL40K (obtainable from Nektar, formerlyShearwater).

Alternative sources of PEG linkers include NOF who supply GL2-400MA3(wherein m in the structure below is 5) and GL2-400MA (where m is 2) andn is approximately 450:

That is to say each PEG is about 20,000 Da.

Thus in one embodiment the PEG is2,3-Bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propyloxy}hexane (the 2 arm branched PEG, —CH2) 3NHCO(CH2)5-MAL, Mw 40,000 knownas SUNBRIGHT GL2-400MA3.

Further alternative PEG effector molecules of the following type:

are available from Dr Reddy, NOF and Jenkem.

In one embodiment there is provided an antibody which is PEGylated (forexample with a PEG described herein), attached through a cysteine aminoacid residue at or about amino acid 229 in the chain, for example aminoacid 229 of the heavy chain (by sequential numbering), for example aminoacid 229 of Seq. ID No. 456, 460, 458, 462, or 464.

In one embodiment the present disclosure provides a Fab′PEG moleculecomprising one or more PEG polymers, for example 1 or 2 polymers such asa 40 kDa polymer or polymers.

Fab′-PEG molecules according to the present disclosure may beparticularly advantageous in that they have a half-life independent ofthe Fc fragment. In one example the present invention provides a methodtreating a disease ameliorated by modulating human aP2 biologicalactivity comprising administering a therapeutically effective amount ofan anti-aP2 antibody or antigen binding agent thereof wherein theantibody or antigen binding agent thereof has a half-life that isindependent of Fc binding to aP2.

In one embodiment there is provided a Fab′ conjugated to a polymer, suchas a PEG molecule, a starch molecule or an albumin molecule.

In one embodiment there is provided a scFv conjugated to a polymer, suchas a PEG molecule, a starch molecule or an albumin molecule.

In one embodiment the antibody or fragment is conjugated to a starchmolecule, for example to increase the half-life. Methods of conjugatingstarch to a protein as described in U.S. Pat. No. 8,017,739 incorporatedherein by reference.

Polynucleotides

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain(s) of an antibody molecule of the presentinvention. Suitably, the DNA sequence encodes the heavy or the lightchain of an antibody molecule of the present invention. The DNA sequenceof the present invention may comprise synthetic DNA, for instanceproduced by chemical processing, cDNA, genomic DNA or any combinationthereof.

DNA sequences, which encode an antibody molecule of the presentinvention, can be obtained by methods well known to those skilled in theart. For example, DNA sequences coding for part or all of the antibodyheavy and light chains may be synthesised as desired from the determinedDNA sequences or on the basis of the corresponding amino acid sequences.

DNA coding for acceptor framework sequences is widely available to thoseskilled in the art and can be readily synthesised on the basis of theirknown amino acid sequences.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibody molecule of the present invention.Desired DNA sequences may be synthesized completely or in part usingoligonucleotide synthesis techniques. Site-directed mutagenesis andpolymerase chain reaction (PCR) techniques may be used as appropriate.

Examples of suitable cDNA sequences are provided in Table 12 below.

Examples of suitable cDNA sequences encoding a humanized light chainvariable region are provided in Seq. ID Nos. 467, 469, 491, 493, 471,and 473. Examples of suitable DNA sequences encoding the humanized heavychain variable region are provided in Seq. ID No. 475, 507, 477, 509,and 511.

Examples of suitable cDNA sequences encoding the light chain (variableand constant) are provided in Seq. ID Nos. 468, 470, 492, 494, 472, and474.

Examples of suitable cDNA sequences encoding the heavy chain (variableand constant) are provided in Seq. ID Nos. 476, 508, 478, 510, and 512.

TABLE 12 Examples of Suitable DNA sequences Encoding anti-aP2 AntibodyFragments Seq. ID cDNA Encoding Identifier No. Sequence Rabbit Ab 909 VLregion (Seq. 465 gacgtcgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagID No. 445) cDNAtcaccatcaagtgccaggccagtgaggatattagtaggtacttagtctggtatcagcagaaaccagggcagcctcccaagcgcctgatctacaaggcatccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacagatttcactctcaccatcagcgacctggagtgtgacgatgctgccacttactactgtcaatgcacttatggtacttatgctggtagttttttttattctttcggcggagggaccgaggtggtcgtcgaa Rabbit Ab 909VH region (Seq. 466cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgac ID No. 454) cDNAactcacctgcacagtctctggattctccctcagtacctactacatgagctgggtccgccaggctccagggaaggggctggaatggatcggaatcatttatcctagtggtagcacatactgcgcgagctgggcgaaaggccgattcaccatctccaaagcctcaaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagacctgataatgatggtacttctggttatttgagtggtttcggcttgtggggccaaggcaccctcgtcaccgtctcgagc 909 gL1 V-region (Seq. ID No. 467gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 446) cDNAgactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagtgtacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatca ag 909 gL1light chain (V + 468gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt constant) (Seq.ID No. 447) gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaaccDNA agaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagtgtacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909 gL10 V-region(Seq. ID No. 469gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 448) cDNAgactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatca ag 909 gL10light chain (V + 470gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt constant) (Seq.ID No. 449) gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaaccDNA agaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909 gL13 V-region(Seq. ID No. 491gacatccaaatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 487) cDNAgactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttctccggcagcggatcgggaaccgagttcactctcaccattagctcactgcagccggaagattttgccacttactactgccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaag 909 gL13 lightchain (V + 492 gacatccaaatgacccagtccccttcctccctttcagccagcgtgggcgatagagtconstant) (Seq. ID No. 489)gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaac cDNAagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttctccggcagcggatcgggaaccgagttcactctcaccattagctcactgcagccggaagattttgccacttactactgccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909 gL50 V-region (Seq.ID No. 493 gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 488)cDNA gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactacgcccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatc aag 909 gL50light chain (V + 494gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt constant) (Seq.ID No. 490) gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaaccDNA agaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactacgcccaggctacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909 gL54 V-region(Seq. ID No. 471gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 450) cDNAgactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagcagacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatc aag 909 gL54light chain (V + 472gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt constant) (Seq.ID No. 451) gactatcacttgccaagcgtcggaggacatctcgcgctcctggtgtggtatcaac cDNAagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagcagacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909 gL55 V-region(Seq. ID No. 473gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt 452) cDNAgactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaacagaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagcatacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatca ag 909 gL55light chain (V + 474gacgtcgtcatgacccagtccccttcctccctttcagccagcgtgggcgatagagt constant) (Seq.ID No. 453) gactatcacttgccaagcgtcggaggacatctcgcgctacctggtgtggtatcaaccDNA agaagccaggtaaagcgcccaagcggctgatctacaaggcctcaactttggcatccggagtgccgtcgaggttcaagggcagcggatcgggaaccgacttcactctcaccattagctcactgcagccggaagattttgccacttactactgccagcatacctacgggacctacgctgggtcgttcttttacagcttcggaggcggaaccaaagtggaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 909gH1 V-region (Seq.ID No. 475 gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc 455)cDNA attgacttgcacggtgagcgggttctcgctttcgacctactacatgtcgtgggtgcgccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcggttcaccatcagcaaggcgtccactactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagc 909 gH1 IgG4 heavy chain (V + 476gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc human γ-4Pconstant) Seq. IDattgacttgcacggtgagcgggttctcgctttcgacctactacatgtcgtgggtgcg No. 456) cDNAccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcggttcaccatcagcaaggcgtccactactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagcgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 909gH14 V-region (Seq. ID No. 507gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc 457) cDNAattgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcgccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcggttcaccatcagcaaggcgtccactaaaaatactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagc 909 gH14 IgG4 heavy chain (V + 508gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc human gamma-4Pconstant) attgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcg(Seq. ID No. 457) cDNAccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcggttcaccatcagcaaggcgtccactaaaaatactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagcgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 909gH15 V-region (Seq. ID. No. 477gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc 459) cDNAattgacttgcacggtgagcgggttctcgctttcgacctactacatgtcgtgggtgcgccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactccgctagctgggccaaggggcggttcaccatcagcaaggcgtccactaaaaatactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgagggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagc 909 gH15 IgG4 heavy chain (V + 478gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc human gamma-4Pconstant) attgacttgcacggtgagcgggttctcgctttcgacctactacatgtcgtgggtgcg(Seq. ID No. 460) cDNAccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactccgctagctgggccaaggggcggttcaccatcagcaaggcgtccactaaaaatactgtggacctcaagctgtcgtcagttactgcggccgacactgcaacctacttttgtgcccgcccggataacgagggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagcgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 909gH61 V-region(Seq. ID. No. 509gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc 461) cDNAattgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcgccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcgggtgaccatcagcaaggactccagcaaaaatcaggtgagcctcaagctgtcgtcagttactgcggccgacactgcagtgtactattgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagc 909 gH61 IgG4 heavy chain (V +510 gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc humangamma-4P constant)attgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcg (Seq. ID No.462) cDNA ccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactgcgctagctgggccaaggggcgggtgaccatcagcaaggactccagcaaaaatcaggtgagcctcaagctgtcgtcagttactgcggccgacactgcagtgtactattgtgcccgcccggataacgatggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagcgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 909gH62 V-region (Seq. ID. No. 511gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc 463) cDNAattgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcgccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactccgctagctgggccaaggggcgggtgaccatcagcaaggactccagcaaaaatcaggtgagcctcaagctgtcgtcagttactgcggccgacactgcagtgtactattgtgcccgcccggataacgagggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagc 909 gH62 IgG4 heavy chain (V +512 gaagtccagctgcaagaatcaggtccaggcctcgtcaaaccatcaggaactttgtc humangamma-4P constant)attgacttgcgccgtgagcgggttctcgctttcgacctactacatgtcgtgggtgcg (Seq. ID No.464) cDNA ccagccgcctgggaagggactggagtggatcggcatcatctacccgtccggcagcacgtactccgctagctgggccaaggggcgggtgaccatcagcaaggactccagcaaaaatcaggtgagcctcaagctgtcgtcagttactgcggccgacactgcagtgtactattgtgcccgcccggataacgagggaacctccggctacctgtccggattcggactgtggggacagggaacccttgtgactgtctcgagcgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag Human IGKV1-17 (A30)-JK4 479gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagt acceptorframework cDNA caccatcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacagcataatagttaccctctcactttcggcggagggaccaaggtggagatcaaa Human IGHV4-4 JH4 acceptor 480caggtgcagctgcaggagtcgggcccaggactggtgaagccttcagggaccct framework cDNAgtccctcacctgcgctgtctctggtggctccatcagcagtagtaactggtggagttgggtccgccagcccccagggaaggggctggagtggattggggaaatctatcatagtgggagcaccaactacaacccgtccctcaagagtcgagtcaccatatcagtagacaagtccaagaaccagttctccctgaagctgagctctgtgaccgccgcggacacggccgtgtattactgtgcgagatactttgactactggggccaaggaaccctggtcacc gtctcctca

The present invention also relates to a cloning or expression vectorcomprising one or more DNA sequences of the present invention.Accordingly, provided is a cloning or expression vector comprising oneor more DNA sequences encoding an antibody of the present invention.Suitably, the cloning or expression vector comprises two DNA sequences,encoding the light chain and the heavy chain of the antibody molecule ofthe present invention, respectively and suitable signal sequences. Inone example the vector comprises an intergenic sequence between theheavy and the light chains (see WO03/048208).

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbour Publishing.

Host Cells Expressing Anti-aP2 Antibodies or Fragments Thereof

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present invention. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present invention. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

Suitable types of Chinese Hamster Ovary (CHO cells) for use in thepresent invention may include CHO and CHO-K1 cells including dhfr− CHOcells, such as CHO-DG44 cells and CHO-DXB11 cells and which may be usedwith a DHFR selectable marker or CHOK1-SV cells which may be used with aglutamine synthetase selectable marker. Other cell types of use inexpressing antibodies include lymphocytic cell lines, e.g., NSO myelomacells and SP2 cells, COS cells. Other suitable cells may include humanembryonic kidney (hek) fibroblasts, for example hek293F and ExpiHekcells, which are known in the art.

In one embodiment, provided is a host cell comprising a cloning orexpression vector comprising a DNA sequence selected from Seq. ID Nos.467, 469, 491, 493, 471, 473, 475, 507, 477, 509, 511, 468, 470, 492,494, 472, 474, 476, 508, 478, 510, or 512.

Production of Anti-aP2 Antibodies or Fragments Thereof

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

There is a provided a process for culturing a host cell and expressingan antibody or fragment thereof, isolating the latter and optionallypurifying the same to provide an isolated antibody or fragment. In oneembodiment the process further comprises the step of conjugating aneffector molecule to the isolated antibody or fragment, for exampleconjugating to a PEG polymer in particular as described herein.

In one embodiment there is provided a process for purifying an antibody(in particular an antibody or fragment according to the invention)comprising the steps: performing anion exchange chromatography innon-binding mode such that the impurities are retained on the column andthe antibody is eluted.

In one embodiment the purification employs affinity capture on a ProteinA column, and then titration. On one embodiment, the purificationemploys affinity capture on a Protein G column, and then HPLC titration.On one embodiment, the purification employs affinity capture on an aP2column, and then titration.

In one embodiment the purification employs cibacron blue or similar forpurification of albumin fusion or conjugate molecules.

Suitable ion exchange resins for use in the process include Q.FF resin(supplied by GE-Healthcare). The step may, for example be performed at apH about 8.

The process may further comprise an initial capture step employingcation exchange chromatography, performed for example at a pH of about 4to 5, such as 4.5. The cation exchange chromatography may, for exampleemploy a resin such as CaptoS resin or SP sepharose FF (supplied byGE-Healthcare). The antibody or fragment can then be eluted from theresin employing an ionic salt solution such as sodium chloride, forexample at a concentration of 200 mM.

Thus the chromatograph step or steps may include one or more washingsteps, as appropriate.

The purification process may also comprise one or more filtration steps,such as a diafiltration step or HPLC filtration step.

Thus in one embodiment there is provided a purified anti-aP2 antibody orfragment, for example a humanised antibody or fragment, in particular anantibody or fragment according to the invention, in substantiallypurified from, in particular free or substantially free of endotoxinand/or host cell protein or DNA.

Purified from as used supra is intended to refer to at least 90% purity,such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

Substantially free of endotoxin is generally intended to refer to anendotoxin content of 1 EU per mg antibody product or less such as 0.5 or0.1 EU per mg product.

Substantially free of host cell protein or DNA is generally intended torefer to host cell protein and/or DNA content 400 μg per mg of antibodyproduct or less such as 100 μg per mg or less, in particular 20 μg permg, as appropriate.

Pharmaceutical Compositions

As the antibodies of the present invention are useful in the treatmentand/or prophylaxis of a pathological condition, the present inventionalso provides a pharmaceutical or diagnostic composition comprising anantibody or antigen binding agent of the present invention incombination with one or more of a pharmaceutically acceptable excipient,diluent, or carrier. Accordingly, provided is the use of an antibody orantigen binding agent of the invention for the manufacture of amedicament. The composition will usually be supplied as part of asterile, pharmaceutical composition that will normally include apharmaceutically acceptable carrier. A pharmaceutical composition of thepresent invention may additionally comprise apharmaceutically-acceptable excipient.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe antibody or antigen binding agent of the present invention togetherwith one or more of a pharmaceutically acceptable excipient, diluent, orcarrier.

The antibody or antigen binding agent may be the sole active ingredientin the pharmaceutical or diagnostic composition or may be accompanied byother active ingredients including other antibody ingredients ornon-antibody ingredients such as steroids or other drug molecules, inparticular drug molecules whose half-life is independent of aP2 binding.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the antibody or antigen binding agent of theinvention. The term “therapeutically effective amount” as used hereinrefers to an amount of a therapeutic agent needed to treat, ameliorate,or prevent a targeted disease or condition, or to exhibit a detectabletherapeutic or preventative effect. For any disclosed antibody orantigen binding agent, the therapeutically effective amount can beestimated initially either in cell culture assays or in animal models,usually in rodents, rabbits, dogs, pigs or primates. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgment of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to500 mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/Kg.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention per dose.

Therapeutic doses of the antibodies or antigen binding agents accordingto the present disclosure show no apparent toxicology effects in vivo.

Advantageously, the levels of aP2 activity in vivo may be maintained atan appropriately reduced level by administration of sequential doses ofthe antibody or binding agent according to the disclosure.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially, orseparately) with other agents, drugs or hormones.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier for administration of the antibody or antigen bindingagent. The carrier should not itself induce the production of antibodiesharmful to the individual receiving the composition and should not betoxic. Suitable carriers may be large, slowly metabolised macromoleculessuch as proteins, polypeptides, liposomes, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymersand inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, it is preferred that the compositions are adapted foradministration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. Direct delivery of the compositions will generally beaccomplished by injection, subcutaneously, intraperitoneally,intravenously or intramuscularly, or delivered to the interstitial spaceof a tissue. The compositions can also be administered into a lesion.Dosage treatment may be a single dose schedule or a multiple doseschedule.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

In one embodiment, the anti-aP2 monoclonal antibodies described hereinare administered as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are described by e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions.

In one embodiment, the anti-aP2 monoclonal antibodies are administeredcontinuously, for example, the antibody can be administered with aneedleless hypodermic injection device, such as the devices disclosedin, e.g., U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413,4,941,880, 4,790,824, or 4,596,556. Examples of implants and modulesuseful in the present invention include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medicants through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known.

Therapeutic Applications

The anti-aP2 monoclonal antibody compounds described herein, includinganti-human aP2 humanized antibody compounds, as well as the disclosedantigen binding agents target the lipid chaperone aP2/FABP4 protein andare useful in treating metabolic disorders, including, but not limitedto, diabetes (for example type 2 diabetes), obesity, and fatty liverdisease, cancer, including liposarcomas, bladder cancer, and ovariancancer, and cardiovascular disorders. It has been surprisinglydiscovered that the anti-aP2 antibody compounds described herein arecapable of binding to secreted aP2 at a low-binding affinity, which,when administered to a host in need thereof, neutralizes the activity ofaP2 and provides lower fasting blood glucose levels, improved systemicglucose metabolism, increased systemic insulin sensitivity, reduced fatmass, liver steatosis, improved serum lipid profiles, and/or reducedatherogenic plaque formation in a host when compared to anti-aP2monoclonal antibodies having higher binding affinities.

In one aspect of the present invention, a method is provided fortreating an aP2 mediated disorder in a host by administering aneffective amount of an anti-aP2 monoclonal antibody or antigen bindingagent described herein. In one embodiment, the disorder is a metabolicdisorder. In one embodiment, the disorder is diabetes. In oneembodiment, the disorder is Type I diabetes. In one embodiment, thedisorder is Type II diabetes. In one embodiment, the disorder ishyperglycemia. In one embodiment, the disorder is obesity. In oneembodiment, the disorder is dyslipidemia. In one embodiment, thedisorder is fatty liver disease. In one embodiment, the disorder is acardiovascular disorder. In one embodiment, the disorder isatherosclerosis. In one embodiment, the disorder is an inflammatorydisorder. In one embodiment, the disorder is asthma. In one embodiment,the disorder is a proliferative disorder, for example, a tumor orneoplasm. In one embodiment, the tumor is selected from transitionalbladder cancer, ovarian cancer, and a liposarcoma. In one embodiment,the disorder is polycystic ovary syndrome (POS).

Metabolic Disorders

In one aspect of the present invention, a method is provided fortreating metabolic disorder in a host by administering an effectiveamount of an anti-aP2 monoclonal antibody described herein. A metabolicdisorder includes a disorder, disease, or condition, which is caused orcharacterized by an abnormal metabolism (i.e., the chemical changes inliving cells by which energy is provided for vital processes andactivities) in a subject. Metabolic disorders include diseases,disorders, or conditions associated with hyperglycemia or aberrantadipose cell (e.g., brown or white adipose cell) phenotype or function.Metabolic disorders can detrimentally affect cellular functions such ascellular proliferation, growth, differentiation, or migration, cellularregulation of homeostasis, inter- or intra-cellular communication;tissue function, such as liver function, renal function, or adipocytefunction; systemic responses in an organism, such as hormonal responses(e.g., insulin response). Examples of metabolic disorders includeobesity, diabetes, hyperphagia, endocrine abnormalities, triglyceridestorage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome,Prader-Labhart-Willi syndrome, and disorders of lipid metabolism.

Diabetes

Diabetes mellitus is the most common metabolic disease worldwide. Everyday, 1700 new cases of diabetes are diagnosed in the United States, andat least one-third of the 16 million Americans with diabetes are unawareof it. Diabetes is the leading cause of blindness, renal failure, andlower limb amputations in adults and is a major risk factor forcardiovascular disease and stroke.

In one aspect of the present invention, a method is provided fortreating diabetes by administering to a host an effective amount of ananti-aP2 monoclonal antibody described herein. In one embodiment, thedisorder is Type I diabetes. In one embodiment, the disorder is Type IIdiabetes.

Type I diabetes results from autoimmune destruction of pancreatic betacells causing insulin deficiency. Type II or non-insulin-dependentdiabetes mellitus (NIDDM) accounts for >90% of cases and ischaracterized by a resistance to insulin action on glucose uptake inperipheral tissues, especially skeletal muscle and adipocytes, impairedinsulin action to inhibit hepatic glucose production, and misregulatedinsulin secretion.

In one embodiment of the present invention, provided herein is a methodof treating Type I diabetes in a host by administering to the host aneffective amount of an anti-aP2 monoclonal antibody described herein incombination or alteration with insulin. In one embodiment of the presentinvention, provided herein is a method of treating Type I diabetes in ahost by administering to the host an effective amount of an anti-aP2monoclonal antibody described herein in combination or alteration with asynthetic insulin analog.

Some people who have Type II diabetes can achieve their target bloodsugar levels with diet and exercise alone, but many also need diabetesmedications or insulin therapy. In one embodiment of the presentinvention, provided herein is a method of treating Type II diabetes in ahost by administering to the host an effective amount of an anti-aP2monoclonal antibody described herein in combination or alteration with acompound selected from metformin (Glucophage, Glumetza); sulfonylureas,including glyburide (DiaBeta, Glynase), glipizide (Glucotrol) andglimepiride (Amaryl); Meglitinides, for example repaglinide (Prandin)and nateglinide (Starlix); thiazolidinediones, for example rosiglitazone(Avandia) and pioglitazone (Actos); DPP-4 inhibitors, for example,sitagliptin (Januvia), saxagliptin (Onglyza) and linagliptin(Tradjenta); GLP-1 receptor agonists, for example Exenatide (Byetta) andliraglutide (Victoza); SGLT2 inhibitors, for example canagliflozin(Invokana) and dapagliflozin (Farxiga); or insulin therapy. Nonlimitingexamples of insulin include Insulin glulisine (Apidra); Insulin lispro(Humalog); Insulin aspart (Novolog); Insulin glargine (Lantus); Insulindetemir (Levemir); Insulin isophane (Humulin N, Novolin N).

In one embodiment, provided herein is a method of treating a disease orcondition associated with diabetes by administering to a host aneffective amount of an anti-aP2 monoclonal antibody described herein.Diseases and conditions associated with diabetes mellitus can include,but are not restricted to, hyperglycemia, hyperinsulinaemia,hyperlipidaemia, insulin resistance, impaired glucose metabolism,obesity, diabetic retinopathy, macular degeneration, cataracts, diabeticnephropathy, glomerulosclerosis, diabetic neuropathy, erectiledysfunction, premenstrual syndrome, vascular restenosis and ulcerativecolitis. Furthermore, diseases and conditions associated with diabetesmellitus comprise, but are not restricted to: coronary heart disease,hypertension, angina pectoris, myocardial infarction, stroke, skin andconnective tissue disorders, foot ulcerations, metabolic acidosis,arthritis, osteoporosis and in particular conditions of impaired glucosetolerance.

Body Weight Disorders

In one embodiment of the present invention, a method is provided fortreating obesity in a host by administering an effective amount of ananti-aP2 monoclonal antibody described herein. Obesity represents themost prevalent of body weight disorders, affecting an estimated 30 to50% of the middle-aged population in the western world.

In one embodiment of the present invention, a method is provided fortreating obesity in a host by administering an effective amount of ananti-aP2 monoclonal antibody or antigen binding agent described hereinin combination or alteration with a second therapeutic agent fortreating obesity. Examples of treatments for obesity include, but arenot limited to phentermine, Belviq (lorcaserin), diethylpropion,phendimetrazine tartrate, Xenical (orlistat), Contrave, orlistat,methamphetamine, Desoxyn, Didrex, Bontril PDM, Suprenza, benzphetamine,Qsymia (phentermine-topiramat), Regimex, naltrexone-bupropion, Evekeo,lorcaserin, and amphetamine sulfate.

In one embodiment, a method is provided for reducing or inhibitingweight gain in a host by administering an effective amount of ananti-aP2 monoclonal antibody or antigen binding agent described herein.

Fatty Liver Disease

There is a need for compositions and methods for the treatment andprevention of the development of fatty liver and conditions stemmingfrom fatty liver, such as nonalcoholic steatohepatitis (NASH), liverinflammation, cirrhosis and liver failure. In one embodiment of thepresent invention, a method is provided for treating fatty liver diseasein a host by administering an effective amount of an anti-aP2 monoclonalantibody or binding agent as described herein.

In one embodiment, the anti-aP2 monoclonal antibody or antigen bindingagent described herein is administered in combination or alteration withomega-3 fatty acids or peroxisome proliferator-activated receptors(PPARs) agonists.

Omega-3 fatty acids are known to reduce serum triglycerides byinhibiting DGAT and by stimulating peroxisomal and mitochondrialbeta-oxidation. Two omega-3 fatty acids, eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), have been found to have high affinity forboth PPAR-alpha and PPAR-gamma. Marine oils, e.g., fish oils, are a goodsource of EPA and DHA, which have been found to regulate lipidmetabolism. Omega-3 fatty acids have been found to have beneficialeffects on the risk factors for cardiovascular diseases, especially mildhypertension, hypertriglyceridemia and on the coagulation factor VIIphospholipid complex activity. Omega-3 fatty acids lower serumtriglycerides, increase serum HDL-cholesterol, lower systolic anddiastolic blood pressure and the pulse rate, and lower the activity ofthe blood coagulation factor VII-phospholipid complex. Further, omega-3fatty acids seem to be well tolerated, without giving rise to any severeside effects. One such form of omega-3 fatty acid is a concentrate ofomega-3, long chain, polyunsaturated fatty acids from fish oilcontaining DHA and EPA and is sold under the trademark Omacor®. Such aform of omega-3 fatty acid is described, for example, in U.S. Pat. Nos.5,502,077, 5,656,667 and 5,698,594, the disclosures of which areincorporated herein by reference.

Peroxisome proliferator-activated receptors (PPARs) are members of thenuclear hormone receptor superfamily ligand-activated transcriptionfactors that are related to retinoid, steroid and thyroid hormonereceptors. There are three distinct PPAR subtypes that are the productsof different genes and are commonly designated PPAR-alpha,PPAR-beta/delta (or merely, delta) and PPAR-gamma. General classes ofpharmacological agents that stimulate peroxisomal activity are known asPPAR agonists, e.g., PPAR-alpha agonists, PPAR-gamma agonists andPPAR-delta agonists. Some pharmacological agents are combinations ofPPAR agonists, such as alpha/gamma agonists, etc., and some otherpharmacological agents have dual agonist/antagonist activity. Fibratessuch as fenofibrate, bezafibrate, clofibrate and gemfibrozil, arePPAR-alpha agonists and are used in patients to decrease lipoproteinsrich in triglycerides, to increase HDL and to decrease atherogenic-denseLDL. Fibrates are typically orally administered to such patients.Fenofibrate or 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-propanoic acid,1-methylethyl ester, has been known for many years as a medicinallyactive principle because of its efficacy in lowering blood triglycerideand cholesterol levels.

Cardiovascular Disease

In one embodiment of the present invention, a method is provided fortreating cardiovascular disease in a host by administering an effectiveamount of an anti-aP2 monoclonal antibody described herein. The anti-aP2antibodies of the present invention are useful in preventing, inhibitingor reducing risk of cardiovascular and cerebrovascular diseasesresulting from atherosclerosis, such as cardiac and/or cerebralischemia, myocardial infarction, angina, peripheral vascular disease andstroke.

In one embodiment, a method is provided for preventing, inhibiting orreducing risk of cardiovascular and cerebrovascular diseases resultingfrom atherosclerosis in a host by administering to the host an effectiveamount of an anti-aP2 monoclonal antibody described herein incombination or alteration with an anti-atherosclerotic agent.

Examples of anti-atherosclerotic agents include, but are not limited to,HMG CoA reductase inhibitors, microsomal triglyceride transfer protein(MTP) inhibitors, fibric acid derivatives, squalene synthetaseinhibitors and other known cholesterol lowering agents, lipoxygenaseinhibitors, ACAT inhibitors, and PPAR α/γ dual agonists as disclosedhereinafter.

The anti-atherosclerotic agent may be an HMG CoA reductase inhibitor,which includes, but is not limited to, mevastatin and related compoundsas disclosed in U.S. Pat. No. 3,983,140, lovastatin (mevinolin) andrelated compounds as disclosed in U.S. Pat. No. 4,231,938, pravastatinand related compounds such as disclosed in U.S. Pat. No. 4,346,227,simvastatin and related compounds as disclosed in U.S. Pat. Nos.4,448,784 and 4,450,171, with pravastatin, lovastatin or simvastatinbeing preferred. Other HMG CoA reductase inhibitors, which may beemployed herein, include, but are not limited to, fluvastatin, disclosedin U.S. Pat. No. 5,354,772, cerivastatin disclosed in U.S. Pat. Nos.5,006,530 and 5,177,080, atorvastatin disclosed in U.S. Pat. Nos.4,681,893, 5,273,995, 5,385,929 and 5,686,104, pyrazole analogs ofmevalonolactone derivatives as disclosed in U.S. Pat. No. 4,613,610,indene analogs of mevalonolactone derivatives as disclosed in PCTapplication WO 86/03488,6-(2-(substituted-pyrrol-1-yl)-alkyl)pyran-2-ones and derivativesthereof as disclosed in U.S. Pat. No. 4,647,576, Searle's SC-45355 (a3-substituted pentanedioic acid derivative) dichloroacetate, imidazoleanalogs of mevalonolactone as disclosed in PCT application WO 86/07054,3-carboxy-2-hydroxy-propane-phosphonic acid derivatives as disclosed inFrench Patent No. 2,596,393, 2,3-disubstituted pyrrole, furan andthiophene derivatives as disclosed in European Patent Application No.0221025, naphthyl analogs of mevalonolactone as disclosed in U.S. Pat.No. 4,686,237, octahydronaphthalenes such as disclosed in U.S. Pat. No.4,499,289, keto analogs of mevinolin (lovastatin) as disclosed inEuropean Patent Application No. 0142146A2, as well as other known HMGCoA reductase inhibitors. In addition, phosphinic acid compounds usefulin inhibiting HMG CoA reductase suitable for use herein are disclosed inGB 2205837.

The squalene synthetase inhibitors suitable for use herein include, butare not limited to, α-phosphono-sulfonates disclosed in U.S. Pat. No.5,712,396, those disclosed by Biller et al, J. Med. Chem., 1988, Vol.31, No. 10, pp 1869-1871, including isoprenoid(phosphinylmethyl)phosphonates as well as other squalene synthetaseinhibitors as disclosed in U.S. Pat. Nos. 4,871,721 and 4,924,024. Inaddition, other squalene synthetase inhibitors suitable for use hereininclude the terpenoid pyrophosphates disclosed by P. Ortiz de Montellanoet al, J. Med. Chem., 1977, 20, 243-249, the farnesyl diphosphate analogA and presqualene pyrophosphate (PSQ-PP) analogs as disclosed by Coreyand Volante, J. Am. Chem. Soc., 1976, 98, 1291-1293,phosphinylphosphonates reported by McClard, R. W. et al, J.A.C.S., 1987,109, 5544 and cyclopropanes reported by Capson, T. L., PhD dissertation,June 1987, Dept. Med. Chem. U of Utah, abstract, Table of Contents, pp16, 17, 40-43, 48-51, Summary.

Other cholesterol lowering drugs suitable for use herein include, butare not limited to, antihyperlipoproteinemic agents such as fibric acidderivatives, such as fenofibrate, gemfibrozil, clofibrate, bezafibrate,ciprofibrate, clinofibrate and the like, probucol, and related compoundsas disclosed in U.S. Pat. No. 3,674,836, probucol and gemfibrozil beingpreferred bile acid sequestrants such as cholestyramine, colestipol andDEAE-Sephadex (Secholex®, Polidexide®), as well as clofibrate,lipostabil (Rhone-Poulenc), Eisal E-5050 (an N-substituted ethanolaminederivatives, imanixii (HOE-402), tetrahydrolipstatin (THL),istigmastanylphosphorylcholine (SPC, Roche), aminocyclodextrin (TanabeSeiyoku), Ajinomoto AJ-814 (azulene derivative), melinamide (Sumitomo),Sandoz 58-035. American Cyanamid CL-277,082 and CL-283,546(disubstituted urea derivatives), nicotinic acid, acipimox, acifran,neomycin, p-aminosalicylic acid, aspirin, poly(diallylmethylamine)derivatives such as disclosed in U.S. Pat. No. 4,759,923, quaternaryamine poly(diallyldimethylarmonium chloride) and ionenes such asdisclosed in U.S. Pat. No. 4,027,009, and other known serum cholesterollowering agents.

The antiatherosclerotic agent may also be a PPAR α/γ dual agonist suchas disclosed by Murakami et al, “A Novel Insulin Sensitizer Acts As aColigand for Peroxisome Proliferator-Activated Receptor Alpha (PPARalpha) and PPAR gamma. Effect on PPAR alpha Activation on Abnormal LipidMetabolism in Liver of Zucker Fatty Rats”, Diabetes 47, 1841-1847(1998).

The anti-atherosclerotic agent may be an ACAT inhibitor such asdisclosed in, “The ACAT inhibitor, Cl-1011 is effective in theprevention and regression of aortic fatty streak area in hamsters”,Nicolosi et al, Atherosclerosis (Shannon, Irel). (1998), 137(1), 77-85;“The pharmacological profile of FCE 27677: a novel ACAT inhibitor withpotent hypolipidemic activity mediated by selective suppression of thehepatic secretion of ApoB100-containing lipoprotein”, Ghiselli,Giancarlo, Cardiovasc. Drug Rev. (1998), 16(1), 16-30; “RP 73163: abioavailable alkylsulfinyl-diphenylimidazole ACAT inhibitor”, Smith, C.,et al, Bioorg. Med. Chem. Lett. (1996), 6(1), 47-50; “ACAT inhibitors:physiologic mechanisms for hypolipidemic and anti-atheroscleroticactivities in experimental animals”, Krause et al, Editor(s): Ruffolo,Robert R., Jr.; Hollinger, Mannfred A., Inflammation: Mediators Pathways(1995), 173-98, Publisher: CRC, Boca Raton, Fla.; “ACAT inhibitors:potential anti-atherosclerotic agents”, Sliskovic et al, Curr. Med.Chem. (1994), 1(3), 204-25; “Inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) as hypocholesterolemic agents. 6. The firstwater-soluble ACAT inhibitor with lipid-regulating activity. Inhibitorsof acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of aseries of substituted N-phenyl-N′-[(1-phenylcyclopentyl)methyl]ureaswith enhanced hypocholesterolemic activity”, Stout et al, Chemtracts:Org. Chem. (1995), 8(6), 359-62.

The other anti-atherosclerotic agent may also be a lipoxygenaseinhibitor including a 15-lipoxygenase (15-LO) inhibitor such asbenzimidazole derivatives as disclosed in WO 97/12615, 15-LO inhibitorsas disclosed in WO 97/12613, isothiazolones as disclosed in WO 96/38144,and 15-LO inhibitors as disclosed by Sendobry et al “Attenuation ofdiet-induced atherosclerosis in rabbits with a highly selective15-lipoxygenase inhibitor lacking significant antioxidant properties,Brit. J. Pharmacology (1997) 120, 1199-1206, and Cornicelli et al,“15-Lipoxygenase and its Inhibition: A Novel Therapeutic Target forVascular Disease”, Current Pharmaceutical Design, 1999, 5, 11-20.

In one embodiment, provided herein is a method of preventing,attenuating or treating a cardiovascular disorder in a host, wherein thehost is peri- or post-menopausal. aP2 is known to increase in peri- andpost-menopausal women, who have a higher incidence of cardiovasculardisease than pre-menopausal women. Accordingly, administering ananti-aP2 monoclonal antibody or antigen binding agent described hereinmay be used to attenuate, prevent, or treat peri- and post-menopausalwomen at risk for developing, or who have developed, cardiovasculardisease associated with elevated levels of circulating aP2.

Inflammatory Disease

The anti-aP2 antibodies and antigen binding agents described herein maybe administered for the treatment of an inflammatory disorder in asubject. Inflammation may arise as a response to an injury or abnormalstimulation caused by a physical, chemical, or biologic agent. Aninflammation reaction may include the local reactions and resultingmorphologic changes, destruction or removal of injurious material suchas an infective organism, and responses that lead to repair and healing.The term “inflammatory” when used in reference to a disorder refers to apathological process, which is caused by, resulting from, or resultingin inflammation that is inappropriate or which does not resolve in thenormal manner. Inflammatory disorders may be systemic or localized toparticular tissues or organs.

Inflammation is known to occur in many disorders which include, but arenot limited to: Systemic Inflammatory Response (SIRS); Alzheimer'sDisease (and associated conditions and symptoms including: chronicneuroinflammation, glial activation; increased microglia; neuriticplaque formation; Amyotrophic Lateral Sclerosis (ALS), arthritis (andassociated conditions and symptoms including, but not limited to: acutejoint inflammation, antigen-induced arthritis, arthritis associated withchronic lymphocytic thyroiditis, collagen-induced arthritis, juvenilearthritis, rheumatoid arthritis, osteoarthritis, prognosis andstreptococcus-induced arthritis, spondyloarthropathies, and goutyarthritis), asthma (and associated conditions and symptoms, including:bronchial asthma; chronic obstructive airway disease, chronicobstructive pulmonary disease, juvenile asthma and occupational asthma);cardiovascular diseases (and associated conditions and symptoms,including atherosclerosis, autoimmune myocarditis, chronic cardiachypoxia, congestive heart failure, coronary artery disease,cardiomyopathy and cardiac cell dysfunction, including: aortic smoothmuscle cell activation, cardiac cell apoptosis and immunomodulation ofcardiac cell function); diabetes (and associated conditions, includingautoimmune diabetes, insulin-dependent (Type I) diabetes, diabeticperiodontitis, diabetic retinopathy, and diabetic nephropathy);gastrointestinal inflammations (and related conditions and symptoms,including celiac disease, associated osteopenia, chronic colitis,Crohn's disease, inflammatory bowel disease and ulcerative colitis);gastric ulcers; hepatic inflammations such as viral and other types ofhepatitis, cholesterol gallstones and hepatic fibrosis; HIV infection(and associated conditions, including—degenerative responses,neurodegenerative responses, and HIV associated Hodgkin's Disease);Kawasaki's Syndrome (and associated diseases and conditions, includingmucocutaneous lymph node syndrome, cervical lymphadenopathy, coronaryartery lesions, edema, fever, increased leukocytes, mild anemia, skinpeeling, rash, conjunctiva redness, thrombocytosis); nephropathies (andassociated diseases and conditions, including diabetic nephropathy,endstage renal disease, acute and chronic glomerulonephritis, acute andchronic interstitial nephritis, lupus nephritis, Goodpasture's syndrome,hemodialysis survival and renal ischemic reperfusion injury);neurodegenerative diseases or neuropathological conditions (andassociated diseases and conditions, including acute neurodegeneration,induction of IL-I in aging and neurodegenerative disease, IL-I inducedplasticity of hypothalamic neurons and chronic stresshyperresponsiveness, myelopathy); ophthalmopathies (and associateddiseases and conditions, including diabetic retinopathy, Graves'ophthalmopathy, inflammation associated with corneal injury or infectionincluding corneal ulceration, and uveitis), osteoporosis (and associateddiseases and conditions, including alveolar, femoral, radial, vertebralor wrist bone loss or fracture incidence, postmenopausal bone loss,fracture incidence or rate of bone loss); otitis media (adult orpediatric); pancreatitis or pancreatic acinitis; periodontal disease(and associated diseases and conditions, including adult, early onsetand diabetic); pulmonary diseases, including chronic lung disease,chronic sinusitis, hyaline membrane disease, hypoxia and pulmonarydisease in SIDS; restenosis of coronary or other vascular grafts;rheumatism including rheumatoid arthritis, rheumatic Aschoff bodies,rheumatic diseases and rheumatic myocarditis; thyroiditis includingchronic lymphocytic thyroiditis; urinary tract infections includingchronic prostatitis, chronic pelvic pain syndrome and urolithiasis;immunological disorders, including autoimmune diseases, such as alopeciaaerata, autoimmune myocarditis, Graves' disease, Graves ophthalmopathy,lichen sclerosis, multiple sclerosis, psoriasis, systemic lupuserythematosus, systemic sclerosis, thyroid diseases (e.g. goitre andstruma lymphomatosa (Hashimoto's thyroiditis, lymphadenoid goitre); lunginjury (acute hemorrhagic lung injury, Goodpasture's syndrome, acuteischemic reperfusion), myocardial dysfunction, caused by occupationaland environmental pollutants (e.g. susceptibility to toxic oil syndromesilicosis), radiation trauma, and efficiency of wound healing responses(e.g. burn or thermal wounds, chronic wounds, surgical wounds and spinalcord injuries), septicaemia, acute phase response (e.g. febrileresponse), general inflammatory response, acute respiratory distressresponse, acute systemic inflammatory response, wound healing, adhesion,immuno-inflammatory response, neuroendocrine response, fever developmentand resistance, acute-phase response, stress response, diseasesusceptibility, repetitive motion stress, tennis elbow, and painmanagement and response.

In one embodiment of the present invention, provided herein is a methodof treating Type I diabetes in a host by administering to the host aneffective amount of an anti-aP2 monoclonal antibody described herein incombination or alteration with an anti-inflammatory agent. Theanti-inflammatory agent can be a steroidal anti-inflammatory agent, anonsteroidal anti-inflammatory agent, or a combination thereof. In someembodiments, anti-inflammatory drugs include, but are not limited to,alclofenac, alclometasone dipropionate, algestone acetonide, alphaamylase, amcinafal, amcinafide, amfenac sodium, amiprilosehydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazidedisodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains,broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen,clobetasol propionate, clobetasone butyrate, clopirac, cloticasonepropionate, cormethasone acetate, cortodoxone, deflazacort, desonide,desoximetasone, dexamethasone dipropionate, diclofenac potassium,diclofenac sodium, diflorasone diacetate, diflumidone sodium,diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide,endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate,felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal,fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid,flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortinbutyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen,fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasolpropionate, halopredone acetate, ibufenac, ibuprofen, ibuprofenaluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacinsodium, indoprofen, indoxole, intrazole, isoflupredone acetate,isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam,loteprednol etabonate, meclofenamate sodium, meclofenamic acid,meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone,methylprednisolone suleptanate, morniflumate, nabumetone, naproxen,naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein,orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride,pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone,piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen,prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazolecitrate, rimexolone, romazarit, salcolex, salnacedin, salsalate,sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac,suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap,tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylicacid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus,pimecorlimus, prodrugs thereof, co-drugs thereof, and combinationsthereof.

Cancers

The invention further provides a method of administering an anti-aP2antibody or antigen binding agent disclosed herein to treat cancer. Inone embodiment, the cancer is selected from liposarcoma, mesenterymixed-mullerian tumor, spinal schwannoma, gallbladder cancer, prostatecancer, brain astrocytoma, lung cancer, ovarian cancer, bladder cancer,colon cancer, esophageal cancer, post-menopausal breast cancer,endometrial cancer, kidney cancer, liver cancer, and pancreatic cancer

In one embodiment of the present invention, a method is provided fortreating a proliferative disorder, for example, a tumor or neoplasm, ina host by administering an effective amount of an anti-aP2 monoclonalantibody or antigen binding agent described herein. In one embodiment,the tumor is selected from transitional bladder cancer, ovarian cancer,and a liposarcoma.

In one embodiment, the cancer is bladder cancer. In one embodiment thecancer is transitional cell carcinoma of the bladder. In one embodimentof the present invention, provided herein is a method of treatingbladder cancer in a host by administering to the host an effectiveamount of an anti-aP2 monoclonal antibody or antigen binding agentdescribed herein.

In one embodiment of the present invention, a method is provided fortreating bladder cancer in a host by administering an effective amountof an anti-aP2 monoclonal antibody or antigen binding agent describedherein in combination or alteration with an additional chemotherapeuticagent. Additional chemotherapeutic agents for treatment of bladdercancer include, but are not limited to, methotrexate, vinblastine,doxorubicin, cisplatin, gemcitabine, carboplatin, paclitaxel, andepirubicin.

In one embodiment the cancer is ovarian cancer. In one embodiment of thepresent invention, provided herein is a method of treating ovariancancer in a host by administering to the host an effective amount of ananti-aP2 monoclonal antibody or an antigen binding agent describedherein.

In one embodiment of the present invention, a method is provided fortreating ovarian cancer in a host by administering an effective amountof an anti-aP2 monoclonal antibody described herein in combination oralteration with an additional chemotherapeutic agent. Additionalchemotherapeutic agents for treatment of ovarian cancer include, but arenot limited to, cisplatin, carboplatin, paclitaxel, docetaxel, albuminbound paclitaxel, altretamine, capecitabine, cyclophosphamide,etoposide, gemcitabine, ifosfamide, irinotecan, liposomal doxorubicin,melphalan, pemetrexed, topotecan, and vinorelbine.

In one embodiment of the present invention, provided herein is a methodof treating liposarcoma in a host by administering an effective amountof an anti-aP2 monoclonal antibody or antigen binding agent describedherein. In one embodiment, the liposarcoma is well-differentiatedliposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, ordedifferentiated liposarcoma. In one embodiment, provided herein is amethod of treating a sarcoma, for example, but not limited to, a fibroushistiocytoma, synovial sarcoma, or leiomyosarcoma. In one embodiment,the anti-aP2 monoclonal antibody or antigen binding agent isadministered in combination with a chemotherapeutic agent and orradiative agent.

In one embodiment, the neoplasm is a benign lipoma, for example, anadenolipoma, angiolipoleiomyoma, angiolipoma, cerebellar pontine angleand internal auditory canal lipoma, chondroid lipoma, corpus callosumlipoma, hibernoma, intradermal spindle cell lipoma, neural fibrolipoma,pleomorphic lipoma, spindle-cell lipoma, and superficial subcutaneouslipoma.

Methods of Attenuating the Severity of an aP2-Mediated Disorder

A method of preventing or treating a disease or disorder caused by anaberrant level of aP2 in a host, typically a human, is provided byadministering to the host a therapeutically effective amount of amonoclonal antibody or antigen binding agent as described herein. Themonoclonal antibody or fragment is administered at a dose sufficient toinhibit or reduce the biological activity of aP2 either partially orfully.

In one aspect, a method of preventing or attenuating the severity of anaP2 mediated disorder in a host is provided by administering aneffective amount of an anti-aP2 monoclonal antibody described herein,resulting in the reduction or attenuation of the biological activity ofsecreted aP2, and a reduction in the associated physiological effects ofelevated aP2 serum levels, for example, a reduction in totalcholesterol, high density lipoprotein (HDL), low density lipoprotein(LDL), very low density lipoprotein (VLDL), and/or triglyceride, fastingblood glucose levels, fat mass levels, hepatic glucose production, fatcell lipolysis, hyperinsulinemia, and/or liver steatosis. In oneembodiment, the attenuation of the biological activity of secreted aP2results in an increase in insulin sensitivity, glucose metabolism,and/or the prevention of islet β-cell death, dysfunction, or loss.

In one aspect of the present invention, a method of reducing totalcholesterol in a host is provided by administering an effective amountof an anti-aP2 monoclonal antibody or antigen binding agent as describedherein. In one embodiment, provided herein is a method of reducing totalcholesterol, high density lipoprotein (HDL), low density lipoprotein(LDL), very low density lipoprotein (VLDL), and/or triglycerides in ahost by administering an effective amount of an anti-aP2 monoclonalantibody described herein.

In other aspects of the present invention, methods are providing for:

reducing fasting blood glucose levels;

reducing fat mass levels;

reducing hepatic glucose production;

reducing fat cell lipolysis;

reducing hyperinsulinemia;

reducing liver steatosis;

increasing glucose metabolism;

increasing insulin sensitivity;

preventing β-cell death, dysfunction, or loss; and/or

determining circulating secreted aP2 levels in a host;

comprising administering an effective amount of an anti-aP2 antibody orantigen binding agent described herein to a host, typically a human, inneed thereof.

EXAMPLES

The lipid chaperone aP2/FABP4 has been implicated in the pathology ofmany immunometabolic diseases, such as diabetes and atherosclerosis.While multiple lines of evidence also support its involvement in humandisease, targeting aP2 for therapeutic applications has not yet beenaccomplished. Recent studies have shown that aP2 is not simply anintracellular protein but also an active adipokine that contributes tohyperglycemia by promoting hepatic gluconeogenesis and interfering withperipheral insulin action. Serum aP2 levels are markedly elevated inmouse and human obesity, and strongly correlate with metaboliccomplications. As an illustrative embodiment, a low binding affinitymonoclonal anti-aP2 antibody CA33, a rabbit-mouse hybrid anti-aP2monoclonal antibody, which includes Rabbit 909 VH (Seq. ID No. 454) and909 VL (Seq. ID No. 445), is described that lowers fasting blood glucoselevels, improves systemic glucose metabolism, increases systemic insulinsensitivity and reduces fat mass and liver steatosis in obese mice. Thestructure of the aP2-CA33 complex was examined and the target epitoperesolved by crystallographic studies in comparison to another monoclonalantibody that lacked efficacy in vivo (anti-aP2 monoclonal antibody H3).In hyperinsulinemic-euglycemic clamp studies, the anti-diabetic effectof CA33 was predominantly linked to the regulation of hepatic glucoseoutput and peripheral glucose utilization. Importantly, this antibodyexhibited no biological effects in aP2-deficient mice, demonstrating itstarget specificity.

Example 1 Preparation of an Illustrative Monoclonal Antibody TargetingSecreted aP2 Animals

Animal care and experimental procedures were performed with approvalfrom animal care committees of Harvard University. Male mice(leptin-deficient (ob/ob) and diet induced obese (DIO) mice withC57BL/6J background) were purchased from The Jackson Laboratory (BarHarbor, Me.) and kept on a 12-hour light/dark cycle. DIO mice withC57BL/6J background were maintained on high-fat diet (60% kcal fat,Research Diets, Inc., D12492i) for 12 to 15 weeks before startingtreatment except in clamp studies, for which they were on HFD for 20weeks. Leptin-deficient (ob/ob) mice were maintained on regular chowdiet (RD, PicoLab 5058 Lab Diet). Animals used were 18 to 31 weeks ofage for dietary models and 9 to 12 weeks of age for the ob/ob model. Inall experiments, at least 7 mice in each group were used, unlessotherwise stated in the text. The mice were treated with 150 μl PBS(vehicle) or 1.5 mg/mouse (˜33 mg/kg) anti-aP2 monoclonal antibody in150 μl PBS by twice a week subcutaneous injections for 3 to 5 weeks(FIG. 1B). Before and after the treatment, blood samples were collectedfrom the tail after 6 hours of daytime food withdrawal. Body weightswere measured weekly in the fed state. Blood glucose levels weremeasured weekly after 6 hours of food withdrawal or after 16 hoursovernight fast. After 2 weeks of treatment, glucose tolerance tests wereperformed by intraperitoneal glucose injections (0.75 g/kg for DIO, 0.5g/kg for ob/ob mice). After 3 weeks of treatment, insulin tolerancetests were performed in DIO mice by intraperitoneal insulin injections(0.75 IU/kg). After 5 weeks of treatment, hyperinsulinemic-euglycemicclamp experiments were performed in DIO mice as previously described(Furuhashi et al., (2007) Nature 447, 959-965; Maeda et al., (2005) Cellmetabolism 1, 107-119).

Metabolic cage (Oxymax, Columbus Instruments) and total body fatmeasurement by dual energy X-ray absorptiometry (DEXA; PIXImus) wereperformed as previously described (Furuhashi et al., (2007) Nature 447,959-965).

Production and Administration of Anti-aP2 Antibodies

CA13, CA15, CA23 and CA33 (Rabbit Ab 909) were produced and purified byUCB. New Zealand White rabbits were immunized with a mixture containingrecombinant human and mouse aP2 (generated in-house in E. coli:accession numbers CAG33184.1 and CAJ18597.1, respectively). Splenocytes,peripheral blood mononuclear cells (PBMCs) and bone marrow wereharvested from immunized rabbits and subsequently stored at −80° C. Bcell cultures from immunized animals were prepared using a methodsimilar to that described by Zubler et al., (“Mutant EL-4 thymoma cellspolyclonally activate murine and human B cells via direct cellinteraction”, J Immunol 134, 3662-3668 (1985)). After a 7 dayincubation, antigen-specific antibody-containing wells were identifiedusing a homogeneous fluorescence-linked immunosorbent assay withbiotinylated mouse or human aP2 immobilized on Superavidin™ beads (BangsLaboratories) and a goat anti-rabbit IgG Fey-specific Cy-5 conjugate(Jackson ImmunoResearch). To identify, isolate and recover theantigen-specific B-cell from the wells of interest, we used thefluorescent foci method (Clargo et al., (2014) mAbs 6, 143-159). Thismethod involved harvesting B cells from a positive well and mixing withparamagnetic streptavidin beads (New England Biolabs) coated withbiotinylated mouse and human aP2 and goat anti-rabbit Fcfragment-specific FITC conjugate (Jackson ImmunoResearch). After staticincubation at 37° C. for 1 h, antigen-specific B cells could beidentified due to the presence of a fluorescent halo surrounding that Bcell. Individual antigen-specific antibody secreting B cells were viewedusing an Olympus IX70 microscope, and were picked with an Eppendorfmicromanipulator and deposited into a PCR tube. Variable region genesfrom these single B-cells were recovered by RT-PCR, using primers thatwere specific to heavy- and light-chain variable regions. Two rounds ofPCR were performed, with the nested 2° PCR incorporating restrictionsites at the 3′ and 5′ ends allowing cloning of the variable region intoa variety of expression vectors; mouse γ1 IgG, mouse Fab, rabbit γ1 IgG(VH) or mouse kappa and rabbit kappa (VL). Heavy- and light-chainconstructs were transfected into HEK-293 cells using Fectin 293(Invitrogen) and recombinant antibody expressed in 6-well plates. After5 days' expression, supernatants were harvested and the antibody wassubjected to further screening by biomolecular interaction analysisusing the BiaCore system to determine affinity and epitope bin.

Mouse anti-aP2 monoclonal antibody H3 was produced by the Dana FarberCancer Institute Antibody Core Facility. Female C57BL/6 aP2−/− mice, 4-6weeks old, were immunized by injection of full-length humanaP2/FABP4-Gst recombinant protein was suspended in Dulbecco's phosphatebuffered saline (PBS; GIBCO, Grand Island, N.Y.) and emulsified with anequal volume of complete Freund's adjuvant (Sigma Chemical Co., St.Louis, Mo.). Spleens were harvested from immunized mice and cellsuspensions were prepared and washed with PBS. The spleen cells werecounted and mixed with SP 2/0 myeloma cells (ATCC No. CRL8-006,Rockville, Md.) that are incapable of secreting either heavy or lightchain immunoglobulins (Kearney et al., (1979) Journal of Immunology 123,1548-1550) at a spleen:myeloma ratio of 2:1. Cells were fused withpolyethylene glycol 1450 (ATCC) in 12 96-well tissue culture plates inHAT selection medium according to standard procedures (Kohler et al.,(1975) Nature 256, 495-497). Between 10 and 21 days after fusion,hybridoma colonies became visible and culture supernatants wereharvested then screened by western blot on strep-His-human-aP2/FABP4. Asecondary screen of 17 selected positive wells was done usinghigh-protein binding 96-well EIA plates (Costar/Corning, Inc. Corning,N.Y.) coated with 50 μl/well of a 2 μg/ml solution (0.1 μg/well) ofstrep-His-human-aP2/FABP4 or an irrelevant Gst-protein and incubatedovernight at 4° C.). Positive hybridomas were subcloned by limitingdilution and screened by ELISA. Supernatant fusions were isotyped withIsostrip kit (RocheDiagnostic Corp., Indianapolis, Ind.).

Large-scale transient transfections were carried out using UCB'sproprietary CHOSXE cell line and electroporation expression platform.Cells were and maintained in logarithmic growth phase in CDCHO media(LifeTech) supplemented with 2 mM Glutamax at 140 rpm in a shakerincubator (Kuhner AG, Birsfelden, Switzerland) supplemented with 8% CO2at 37° C. Prior to transfection, the cell numbers and viability weredetermined using CEDEX cell counter (Innovatis AG. Bielefeld, Germany)and 2×108 cells/ml were centrifuged at 1400 rpm for 10 minutes. Thepelleted cells were washed in Hyclone MaxCyte buffer (Thermo Scientific)and respun for a further 10 minutes and the pellets were re-suspended at2×108 cells/ml in fresh buffer. Plasmid DNA, purified using QIAGENPlasmid Plus Giga Kit® was then added at 400 μg/ml. Followingelectroporation using a MAxcyte STX® flow electroporation instrument,the cells were transferred in ProCHO medium (Lonza) containing 2 mMGlutamax and antibiotic antimitotic solution and cultured in wave bag(Cell Bag, GE Healthcare) placed on Bioreactor platform set at 37° C.and 5% CO2 with wave motion induced by 25 rpm rocking.

Twenty-four hours post transfection a bolus feed was added and thetemperature was reduced to 320C and maintained for the duration of theculture period (12-14 days). At day 4 3 mM sodium butryrate (n-BUTRICACID sodium salt, Sigma B-5887) was added to the culture. At day 14, thecultures were centrifuged for 30 minutes at 4000 rpm and the retainedsupernatants were filtered through 0.22 μm SARTO BRAN-P (Millipore)followed by 0.22 μm Gamma gold filters. CHOSXE harvest expressing mousemonoclonal antibody (mAb) was conditioned by addition of NaCl (to 4M).The sample was loaded onto a protein A MabSelect Sure packed column(GE-healthcare) equilibrated with 0.1M Glycine+4M NaCl pH8.5 at 15ml/min. The sample was washed with 0.1M Glycine+4M NaCl pH8.5 and anadditional wash step was performed with 0.15M Na2HPO4 pH 9. The U.Vabsorbance peak at A280 nm was collected during elution from the columnusing 0.1M sodium citrate pH 3.4 elution buffer and then neutralized topH 7.4 by addition of 2M Tris-HCl pH 8.5. The mAb pool from protein Awas then concentrated to suitable volume using a minisette TangentialFlow Filtration device before being purified further on a HiLoad XK50/60 Superdex 200 prep grade gel filtration column (GE-healthcare).Fractions collected were then analysed by analytical gel filtrationtechnique for monomer peak and clean monomer fractions pooled as finalproduct. The final product sample was then characterised by reducing andnon-reduced SDS-PAGE and analytical gel filtration for final puritycheck. The sample was also tested and found to be negative for endotoxinusing a LAL assay method for endotoxin measurements. The final bufferfor all mAbs tested was PBS. For in vivo analysis, purified antibodieswere diluted in saline to 10 mg/ml and injected at a dose of 1.5mg/mouse (33 mg/kg) into ob/ob and WT mice on high-fat diet.

Measurement of Antibody Affinity

The affinity of anti-aP2 binding to aP2 (recombinantly generated in E.coli as described below) was determined by biomolecular interactionanalysis, using a Biacore T200 system (GE Healthcare). AffinipureF(ab′)2 fragment goat anti-mouse IgG, Fc fragment specific (JacksonImmunoResearch Lab, Inc.) in 10 mM NaAc, pH 5 buffer was immobilized ona CM5 Sensor Chip via amine coupling chemistry to a capture levelbetween 4500-6000 response units (RU) using HBS-EP+ (GE Healthcare) asthe running buffer. Anti-aP2 IgG was diluted to between 1-10 μg/ml inrunning buffer. A 60 s injection of anti-aP2 IgG at 10 μl/min was usedfor capture by the immobilized anti-mouse IgG, Fc then aP2 was titratedfrom 25 nM to 3.13 nM over the captured anti-aP2 for 180 s at 30 μl/minfollowed by 300 s dissociation. The surface was regenerated by 2×60 s 40mM HCl and 1×30 s 5 mM NaOH at 10 μl/min. The data were analyzed usingBiacore T200 evaluation software (version 1.0) using the 1:1 bindingmodel with local Rmax. For CA33, 60 s injection of the antibody at 10μl/min was used for capture by the immobilized anti-mouse IgG, Fc thenaP2 was titrated from 40 μM to 0.625 μM over the captured anti-aP2 for180 s at 30 μl/min followed by 300 s dissociation. The surface wasregenerated by 1×60 s 40 mM HCl, 1×30 s 5 mM NaOH and 1×60 s 40 mM HClat 10 μl/min. Steady state fitting was used to determine affinityvalues.

Antibody Cross-Blocking

The assay was performed by injecting mouse aP2 in the presence orabsence of mouse anti-aP2 IgG over captured rabbit anti-aP2 IgG.Biomolecular interaction analysis was performed using a Biacore T200 (GEHealthcare Bio-Sciences AB). Anti-aP2 rabbit IgG transient supernatantswere captured on the immobilized anti-rabbit Fc surfaces (onesupernatant per flowcell) using a flow rate of 10 μl/min and a 60 sinjection to give response levels above 200RU. Then mouse aP2 at 100 nM,0 nM or mouse aP2 at 100 nM plus mouse anti-aP2 IgG at 500 nM werepassed over for 120 s followed by 120 s dissociation. The surfaces wereregenerated with 2×60 s 40 mM HCl and 1×30 s 5 mM NaOH.

FABP Cross-Reactivity

The recombinant human proteins aP2 (generated at UCB in E. coli (seemethod below)), hFABP3 (Sino Biological Inc.) and hFABP5/hMal1 (SinoBiological Inc.) were biotinylated in a 5-fold molar excess of EZ-Link®Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) and purified from unboundbiotin using a Zeba desalting column (Thermo Fisher Scientific). Allbinding studies were performed at 25° C. using a ForteBio Octet RED384system (Pall ForteBio Corp.). After a 120 s baseline step in PBScontaining 0.05% Tween 20, pH7.4 (PBS-T), Dip and Read™ streptavidin(SA) biosensors (Pall ForteBio Corp.) were loaded with biotinylatedrecombinant haP2, hFABP3 or hFABP5/hMal1 at 60 nM for 90 s. After a 60 sstabilisation step in PBS-T, each FABP-loaded biosensor was transferredto a sample of monoclonal antibody at 800 nM and association wasmeasured for 5 min. Biosensors were then transferred back to PBS-T for 5min to measure dissociation. Non-specific binding of antibodies wasmonitored using unloaded biosensor tips. Maximal association bindingi.e., once signal had plateaued, minus background binding, was plottedfor each antibody/FABP combination.

aP2 Expression and Purification

Mouse (or human) aP2 cDNA optimized for expression in E. coli waspurchased from DNA 2.0 (Menlo Park, Calif.) and subcloned directly intoa modified pET28a vector (Novagen) containing an in-frame N-terminal 10His-tag followed by a Tobacco Etch Virus (TEV) protease site. Proteinwas expressed in the E. coli strain BL21DE3 and purified as follows.Typically, cells were lysed with a cooled cell disruptor (ConstantSystems Ltd.) in 50 ml lysis buffer (PBS (pH 7.4) containing 20 mMimidazole) per liter of E. coli culture supplemented with a Completeprotease inhibitor cocktail tablet, EDTA-free (Roche, Burgess Hill).Lysate was then clarified by high-speed centrifugation (60000 g, 30minutes, 4° C.). 4 ml/Ni-NTA beads (Qiagen) were added per 100 mlcleared lysate and tumbled for 1 h at 4° C. Beads were packed in aTri-Corn column (GE Healthcare) attached to an AKTA FPLC (GE LifeSciences) and protein eluted in a buffer containing 250 mM imidazole.Fractions containing protein of interest as judged by 4-12% Bis/TrisNuPage (Life Technologies Ltd.) gel electrophoresis were dialyzed toremove imidazole and treated with TEV protease at a ratio of 1 mg per100 mg protein. After overnight incubation at 4° C. the sample wasre-passed over the Ni/NTA beads in the Tri-Corn column. Untagged (i.e.TEV cleaved) aP2 protein did not bind to the beads and was collected inthe column flow through. The protein was concentrated, and loaded ontoan S75 26/60 gel filtration column (GE healthcare) pre-equilibrated inPBS, 1 mM DTT. Peak fractions were pooled and concentrated to 5 mg/ml.Six ml of this protein was then extracted and precipitated withacetonitrile at a ratio of 2:1 to remove any lipid. Followingcentrifugation at 16000 g for 15 mins the acetonitrile+buffer wasremoved for analysis of original lipid content. The pellet of denaturedprotein was then resuspended in 6 ml of 6 M GuHCl PBS+2 μMoles palmiticacid (5:1 ratio of palmitic acid to aP2) and then dialyzed two timesagainst 5 L PBS for 20 hrs at 4° C. to allow refolding. Followingcentrifugation to remove precipitate (16000 g, 15 minutes) protein wasgel filtered using a S75 26/20 column in PBS to remove aggregate. Peakfractions were pooled and concentrated to 13 mg/ml.

aP2 Crystallography

Purified mouse aP2 was complexed with CA33 and H3 Fab (generated at UCBby conventional methods) as follows. Complex was made by mixing 0.5 mlof aP2 at 13 mg/ml with either 0.8 ml of CA33 Fab at 21.8 mg/ml or 1.26ml of H3 Fab at 13.6 mg/ml (aP2:Fab molar ratio of 1.2:1). Proteins wereincubated at RT for 30 minutes then run on an S75 16/60 gel filtrationcolumn (GE Healthcare) in 50 mM Tris pH7.2, 150 mM NaCl+5% glycerol.Peak fractions were pooled and concentrated to 10 mg/ml forcrystallography.

Sitting-drop crystallization trials were set up using commerciallyavailable screening kits (QIAGEN). Diffraction-quality crystals wereobtained directly in primary crystallization screening without any needto optimize crystallization conditions. For the aP2/CA33 complex thewell solution contained 0.1 M Hepes pH 7.5, 0.2 M (NH₄)₂SO₄, 16% PEG 4Kand 10% isopropanol. For the aP2/H3 complex the well solution contained0.1 M MES pH5.5, 0.15 M (NH₄)₂SO₄ and 24% PEG 4K. Data were collected atthe Diamond Synchrotron on i02 (λ=0.97949) giving a 2.9 Å dataset foraP2/CA33 and a 2.3 Å dataset for aP2/H3. Structures were determined bymolecular replacement using Phaser (44) (CCP4) with AP2 and a Fab domainstarting models. Two complexes were found to be in the asymmetric unitfor aP2/CA33 and one for aP2/H3. Cycles of refinement and model buildingwere performed using CNS (Brunger et al., (2007) Nature Protocols 2,2728-2733) and coot (Emsley et al., (2004) Acta crystallographica.Section D. Biological crystallography 60, 2126-2132) (CCP4) until allthe refinement statistics converged for both models. Epitope informationdescribed above was derived by considering atoms within 4 Å distance atthe aP2/Fab contact surface. The data collection and refinementstatistics are shown below. Values in parenthesis refer to the highresolution shell.

Structure aP2-CA33 aP2-H3 Space group P 1 2₁ 1 P 1 2₁ 1 Cell dimensionsa, b, c (Å) 65.27, 101.95, 95.31 71.50, 66.04, 75.68 α, β, γ (°) 90.00,90.03, 90.00 90.00, 111.67, 90.00 Resolution (Å) 54.97-2.95 (3.09-2.95) 33.03-2.23 (2.37-2.23)  R_(sym) or R_(merge)  0.18 (1.169)  0.11 (0.352)I/σI 8.3 (2.9) 6.8 (1.7) Completeness (%) 99.2 (98.9) 98.6 (98.4)Redundancy 6.2 (6.3) 2.6 (2.6) Refinement Resolution (Å) 54.97-2.9533.02-3.00 No. reflections 24898 13077 R_(work)/R_(free) 0.21/0.280.22/0.27 No. atoms Protein 8632 4331 Water 0 0 B-factors aP2(molecule 1) 58.3; 27.5 (molecule 2) 64.6 Fab (molecule 1) 52.9; 22.5(molecule 2) 52.5 R.m.s. deviations Bond lengths (Å) 0.009 0.011 Bondangles (°) 1.42 1.67 Values in parenthesis refer to the high resolutionshell. R_(sym) = Σ|(I − <I>)|/Σ(I), where I is the observed Integratedintensity, <I> is the average integrated intensity obtained frommultiple measurements, and the summation is over all observedreflections. R_(work) = Σ||F_(obs)| − k|F_(calc)||/Σ|F_(obs)|, whereF_(obs) and F_(calc) are the observed and calculated structure factors,respectively. R_(free) is calculated as R_(work) using 5% of thereflection data chosen randomly and omitted from the refinementcalculations. Epitope information was derived by considering atomswithin 4 Å distance at the aP2/Fab contact surface.

Hyperinsulinemic-Euglycemic Clamp Studies and Hepatic Biochemical Assays

Hyperinsulinemic-euglycemic clamps were performed by a modification of areported procedure (Cao et al., (2013) Cell Metab. 17, 768-778).Specifically, mice were clamped after 5 hours fasting and infused with 5mU/kg/min insulin. Blood samples were collected at 10-min intervals forthe immediate measurement of plasma glucose concentration, and 25%glucose was infused at variable rates to maintain plasma glucose atbasal concentrations. Baseline whole-body glucose disposal was estimatedwith a continuous infusion of [3-3H]-glucose (0.05 μCi/min). This wasfollowed by determination of insulin-stimulated whole-body glucosedisposal whereby [3-3H]-glucose was infused at 0.1 μCi/min.

Total lipids in liver were extracted according to the Bligh-Dyerprotocol (Bligh et al., (1959) Canadian J. Biochem. and Phys. 37,911-917), and a colorimetric method used for triglyceride contentmeasurement by a commercial kit according to manufacturer's instructions(Sigma Aldrich). Gluconeogenic enzyme Pck1 activity was measured by amodification of reported method (Petrescu et al., (1979) AnalyticalBiochem. 96, 279-281). Glucose-6-phosphatase (G6pc) activity wasmeasured by a modification of Sigma protocol [EC 3.1.3.9]. Briefly, thelivers were homogenized in lysis buffer containing 250 mM sucrose, TrisHCl and EDTA. Lysates were centrifuged at full speed for 15 min and thesupernatant (predominantly cytoplasm) isolated. Then microsomalfractions were isolated by ultracentrifugation of cytoplasmic samples.Microsomal protein concentrations were measured by commercial BCA kit(Thermo Scientific Pierce). 200 mM glucose-6-phosphate (Sigma Aldrich)was pre-incubated in Bis-Tris. 150 μg microsomal protein or serialdilution of recombinant G6Pase were added and incubated in that solutionfor 20 min at 37° C. Then 20% TCA was added, mixed and incubated for 5min at room temperature. Samples were centrifuged at full speed at 4° C.for 10 min, and the supernatant was transferred to a separate UV plate.Color reagent was added and absorbance at 660 nm was measured andnormalized to standard curve prepared with serial dilution ofrecombinant glucose-6-phosphatase (G6pc) enzyme.

Plasma aP2, mal1, FABP3, Adiponectin, Glucagon, and Insulin ELISAs

Blood was collected from mice by tail bleeding after 6 hours daytime or16 hours overnight food withdrawal. Terminal blood samples werecollected by cardiac puncture or collected from tail vein. The sampleswere spun in a microcentrifuge at 3,000 rpm for 15 minutes at 4° C.Plasma aP2 (Biovendor R&D), mal1 (Circulex Mouse Mal1 ELISA, CycLex Co.,Ltd., Japan), FABP3 (Hycult Biotech, Plymouth Meeting, Pa.) glucagon,adiponectin (Quantikine ELISA, R&D Systems, Minneapolis, Minn.), andinsulin (insulin-mouse ultrasensitive ELISA, Alpco Diagnostics, Salem,N.H.) measurements were performed according to the manufacturer'sinstructions.

Quantitative Real Time PCR Analysis

Tissues were collected after 6 hours daytime food withdrawal,immediately frozen and stored at −80° C. RNA isolation was performedusing Trizol (Invitrogen, Carlsbad, Calif.) according to themanufacturer's protocol. For first strand cDNA synthesis 0.5-1 ng RNAand 5× iScript RT Supermix were used (BioRad Laboratories, Herculus,Calif.). Quantitative real time PCR (Q-PCR) was performed using PowerSYBR Green PCR master mix (Applied Biosystems, Life Technologies, GrandIsland, N.Y.) and samples were analyzed using a ViiA7 PCR machine(Applied Biosystems, Life Technologies, Grand Island, N.Y.). Primersused for Q-PCR were as follows:

36B4 5′-cactggtctaggacccgagaa-3′ Seq. ID No. 513;5′-agggggagatgttcagcatgt-3′ Seq. ID No. 514 FAS 5′-ggaggtggtgatagccggtat-3′ Seq. ID No. 515; 5′-tgggtaatccatagagcccag-3′ Seq. ID No. 516SCD1 5′-ttcttgcgatacactctggtgc-3′ Seq. ID No. 517;5′-cgggattgaatgacttgtcgt-3′ Seq. ID No. 518 Pck15′-ctgcataacggtctggacttc-3′ Seq. ID No. 519;5′-cagcaactgcccgtactcc-3′ Seq. ID No. 520 G6pc5′-cgactcgctatctccaagtga-3′ Seq. ID No. 521;5′-gttgaaccagtctccgacca-3′ Seq. ID No. 522 ACC15′-atgtctggcttgcacctagta-3′ Seq. ID No. 523;5′-ccccaaagcgagtaacaaattct-3′ Seq. ID No. 524 TNF5′-ccctcacactcagatcatcttct-3′ Seq. ID No. 525; 5′-gctacgacgtgggctacag-3′ Seq. ID No. 526 IL-1β 5′-gcaactgttcctgaactcaact-3′ Seq. ID No.527; 5′-atcttttggggtccgtcaact-3′ Seq. ID No. 528 IL-6 5′-acaaccacggccttccctactt-3′ Seq. ID No. 529; 5′-cacgatttcccagagaacatgtg-3′ Seq.ID No. 530 CCL2 5′-catccacgtgttggctca-3′ Seq. ID No. 531;5′-gatcatcttgctggtgaatgagt-3′ Seq. ID No. 532 CXCL15′-gactccagccacactccaac-3′ Seq. ID No. 533;5′-tgacagcgcagctcattg-3′ Seq. ID No. 534 F4/805′-tgactcaccttgtggtcctaa-3′ Seq. ID No. 535;5′-cttcccagaatccagtctttcc-3′ Seq. ID No. 536 CD685′-tgtctgatcttgctaggaccg-3′ Seq. ID No. 537;5′-gagagtaacggccatttttgtga-3′ Seq. ID No. 538 TBP5′-agaacaatccagactagcagca-3′ Seq. ID No. 539;5′-gggaacttcacatcacagctc-3′ Seq. ID No. 540

Statistical Analysis

Results are presented as the mean±SEM. Statistical significance wasdetermined by repeated measures ANOVA or student's t test. * denotessignificance at p<0.05, **denotes significance at p<0.01.

Anti-aP2 Monoclonal Antibody Development and Screening

Obesity is associated with increased levels of circulating aP2, whichcontributes to the elevation of hepatic glucose production and reducedperipheral glucose disposal and insulin resistance, characteristics oftype 2 diabetes. Therefore, neutralizing serum aP2 represents anefficient approach to treat diabetes and possibly other metabolicdiseases.

Mouse and rabbit-mouse hybrid monoclonal antibodies raised against thehuman and mouse aP2 peptides were produced and screened. Assessment ofbinding affinity by biomolecular interaction analysis using a Biacoresystem demonstrated a wide range of affinities for these antibodies,from the micromolar to the low nanomolar range (FIG. 1A). As an initialtest for the potential effects of these antibodies in vivo, theantibodies were administered subcutaneously for 4 weeks to mice withhigh fat diet (HFD)-induced obesity (FIG. 1B). The HFD-feeding resultedin a rise in serum insulin levels during the experiment, an effect thatwas blunted by treatment with the mouse antibody H3 and reversed by thehybrid antibody CA33, but unaltered by the other three hybrid antibodiestested (FIG. 1C). Interestingly, CA33 also significantly decreasedfasting blood glucose (FIG. 1D), while the other antibodies tested didnot improve glycemia, indicating that CA33 reduced insulin resistanceassociated with HFD and improved glucose metabolism. The systemicimprovement in glucose metabolism was further verified using a glucosetolerance test (GTT). CA33 therapy resulted in significantly improvedglucose tolerance (FIG. 1E), while the other antibodies did not improveglucose tolerance and glucose disposal curves were not differentcompared to vehicle (FIG. 7A). Furthermore, only CA33 treatment markedlyimproved insulin sensitivity as demonstrated in insulin tolerance tests,while other antibodies tested were similar to vehicle (FIG. 1F, FIG.7B). There was a moderate reduction in weight gain in all but one of theantibody-treated groups (CA15) (FIG. 1G), although this did notcorrelate with improvement in glucose metabolism. Taken together, theseresults demonstrated that CA33 uniquely possessed anti-diabeticproperties.

CA33 is a Low-Affinity Antibody that Neutralizes aP2

CA33 was further characterized to better understand its uniquetherapeutic properties. In an octet-binding assay, all of the antibodiestested demonstrated saturable binding to aP2. There was a measurable butlow interaction with the related protein FABP3 (˜25% of the aP2/FABP4interaction), and only minor interaction with Mal1/FABP5 that wassimilar to control IgG (FIG. 2A). Interestingly, we also found that theimprovement in glucose homeostasis in CA33-treated mice was related to aunique effect of this antibody on circulating aP2 levels. After 4 weeksof treatment, CA33 treated mice maintained circulating aP2 levels at alevel similar to or slightly lower than that seen in control-treatedanimals, while all other antibodies including H3 resulted in a dramatic10-fold increase in circulating aP2 levels (FIG. 2B). Indeed,circulating aP2 was undetectable by western blot in both control andCA-33-treated mice, but robustly evident in serum following H3 treatment(FIG. 2B, inset).

In cross blocking experiments to begin characterizing the target sites,we found that CA33 partially blocked binding of the ineffective mouseantibody H3 to aP2, while H3 binding was completely blocked by thehybrid antibodies CA13 and CA15 (FIG. 2C). These data suggest that theepitope recognized by CA33 only partially overlaps with that recognizedby H3. In further analysis, epitope identification based onhydrogen-deuterium exchange mass spectrometry experiments, for example,as described by Pandit et al. (2012) J. Mol. Recognit. March;25(3):114-24 (incorporated herein by reference), indicated interactionof CA33 with first alpha helix and the first beta sheet of aP2 onresidues 9-17, 20-28 and 118-132, which partially overlapped with theepitope identified for H3 (FIG. 2D). To understand these epitopesprecisely, we then co-crystallized the Fab fragments of CA33 and H3 withaP2 (FIG. 2E). Analyses of the crystals showed that CA33 binds anepitope spread out over the secondary structure elements beta1 andbeta10 and the random coil regions linking alpha2 to beta2 and beta3 tobeta4, and includes the aP2 amino acids 57T, 38K, 11L, 12V, 10K and 130E(FIG. 2F). Despite the partial blocking of H3 by CA33, we observed thatthere is in fact no direct overlap of their epitopes. Instead, thesignificant movement of the region around aP2 Phe58 may partially blockbinding of one antibody by the other in the competition experiments. Inaddition, the low affinity of the CA33 Fab can be explained by thecrystal structure. Unusually, only one amino acid in the heavy chain ofCA33 makes a contact with aP2, and the majority of the contacts arethrough the light chain (FIG. 2E, F). In contrast, H3-aP2 contact ismore conventional, with both Fab chains interacting with aP2. Thestructure also shows that CA33 does not bind to the ‘lid’ of theβ-barrel (14S to 37A), which has been postulated to control access oflipids to the binding pocket or the ‘hinge’ which contains E15, N16, andF17. In addition, it was found that lipid binding (paranaric acid) toaP2 was not substantially altered by the presence of CA33 (FIG. 2G). H3does bind directly to the ‘lid’ but has limited activity. Binding ofCA33 to lipid-bound aP2 or lipid-free aP2 was also examined usingbiochemical analysis (Biacore). As seen in FIGS. 2H and 2I, CA33 bindsto both lipid-bound aP2 and lipid-free aP2 with the followingaffinities:

Mouse aP2 KD Lipid-loaded 9.3 μM De-lipidated 4.7 μMCumulatively, these results suggest that CA33 activity may beindependent of aP2 lipid binding.

Given the relatively low affinity of CA33 for aP2, off-target effectswere examined. The effect of CA33 treatment in aP2−/− mice fed a HFDwere tested. In these experiments, antibody therapy failed to induce anychange in weight or fasting glucose in this model (FIG. 3A, 3B).Furthermore, CA33 did not affect glucose tolerance in obese aP2−/− mice(FIG. 3C), clearly demonstrating that the antibody's effects are due totargeting aP2.

Finally, the effect of CA33 in a second model of severe genetic obesityand insulin resistance using leptin-deficient ob/ob mice was examined.Over the course of 3 weeks of treatment, both the CA33 andvehicle-treated groups gained weight but the extent was less in theCA33-treated group (FIG. 3D). Strikingly, hyperglycemia in the ob/obmice was normalized in CA33-treated mice compared to controls (FIG. 3E).The extent of hyperinsulinemia was also partially reduced in theCA33-treated animals (FIG. 3F). Normal glucose and lower insulin levelssuggest improved glucose metabolism upon neutralization of aP2. Indeed,following administration of exogenous glucose, CA33 treated ob/ob micealso exhibited significantly improved glucose tolerance compared tovehicle treated mice despite the presence of massive obesity (FIG. 3G).These data underscore the broad applicability of aP2 neutralization tometabolic disease in independent preclinical models and are consistentwith the results obtained in the context of dietary and genetic obesity.

CA33 Treatment Improves Lipid Metabolism and Inhibits Hepatosteatosis inObese Mice

Having identified and physically characterized a candidate monoclonalantibody that could generate metabolic benefits, detailed functionalstudies in the HFD model were performed. To further explore themetabolic effects of CA33 treatment, we treated HFD-induced obese micewith CA33 or vehicle for five weeks and examined the effects on liver.As expected, long term high fat feeding induced steatosis andtriglyceride accumulation in vehicle-treated mice, however, theseeffects were significantly ameliorated by CA33 treatment (FIG. 4A, B).In addition, the improvement in liver lipid homeostasis was accompaniedby reduced hepatic expression of key genes involved in de novolipogenesis (FIG. 4C). Similar to genetic aP2 deficiency, CA33 treatedmice had moderately higher plasma free fatty acid levels (FIG. 4D) andlower glycerol levels (FIG. 4E). Total cholesterol levels were alsolower in CA33 treated group (FIG. 4F), although there was no significantdifference in plasma triglycerides (FIG. 4G). Notably, although completewhole body genetic aP2 deficiency is associated with a substantialupregulation of tissue mal1 (FABP5/Mal1) expression, significant changesin circulating mal1 levels upon CA33 treatment indicating lack ofcompensatory changes were not detected (FIG. 4H). In addition, CA33treatment did not significantly alter the circulating levels of FABP3protein (FIG. 4H). There was also no alteration in the serum levels ofglucagon or adiponectin in CA33 treated mice (FIG. 4I-J).

Effects of CA33 Treatment on Adipose Tissue and Body Composition

The impact of CA33 treatment on adipose tissue and body composition wasalso examined. Dual-energy X-ray absorptiometry (DEXA) scans showed thatCA33-treatment significantly reduced fat mass (FIG. 5A); while lean masswas also slightly reduced, this likely reflects the reduced lipidaccumulation in solid organs, especially liver (FIG. 4A, B). Indeed,CA33 treatment significantly decreased liver weight, and this reductionremained significant when expressed as percentage of body weight (FIG.5B). The decrease in body weight in CA33-treated obese mice isreminiscent of the phenotype of mice with aP2-deficiency and combinedaP2/mal1-deficiency on HFD, and raised the possibility that the antibodytherapy directly altered metabolic parameters. In metabolic cageanalysis, physical activity, food intake, and heat production weresimilar in vehicle and antibody-treated mice (FIG. 5C, D).

CA33 May Increase Fatty Acid Oxidation

For additional metabolic measurements, obese mice fed a HFD were placedin an indirect open circuit calorimeter (Oxymax System, ColumbusInstruments). Oxygen and carbon dioxide concentrations by volume weremonitored at the inlet and outlet parts of a partially sealed chamber,through which a known flow of ambient air was forcibly ventilated. Theconcentration difference measured between the parts was used to computeoxygen consumption (VO2) and carbon dioxide production (VCO2), and tocalculate respiratory exchange rate (VCO2/VO2). In metabolic cageanalysis, obese mice who were fed HFD and treated with CA33 for eightweeks showed increased VO2 Utilization compared to vehicle treated mice(FIG. 5E), resulting in a respiratory exchange ratio (RER) closer to 0.7(FIG. 5F), suggesting that the antibody increased lipid utilization.

Histological analysis revealed that, as expected, prolonged HFD-feedingresulted in abundant lipid accumulation in brown adipose tissue (BAT).However, treatment with CA33 resulted in a significant decrease in BATtissue weight and dramatically decreased lipid droplet size (FIG. 5G,5H).

In general, adipose tissue depots appeared similar betweenantibody-treated and control groups; although there was a significantdecrease in the size of the perigonadal fat pad (PGWAT; FIG. 5I).Analysis of H&E stained sections of PGWAT revealed a similar degree ofinflammatory cell infiltration in CA33 and vehicle-treated mice. (FIG.5J). Indeed, the infiltration of F4/80 and CD11b positive myeloid cellsin adipose tissue preparations by FACS analysis indicated a similarnumber of these immune cells between the groups (FIG. 5K, L). Inaddition, there was no change observed in the expression of theinflammatory markers TNF, IL-1β, CCL2, CXCL1, F4/80 or CD68 inperigonadal white adipose tissue (PG-WAT) isolated from CA33-treatedmice and only a modest increase in IL-6 mRNA levels (FIG. 5M). Adiposetissue aP2 protein levels showed a slight increase upon antibodytreatment (FIG. 5N and FIG. 5P), and we did not observe compensatoryregulation of mal1 (FIG. 5O and FIG. 5P), suggesting that neutralizingcirculating aP2 in adult mice does not result in molecular compensationby other FABP isoforms, and in this way differs from genetic deficiency.

CA33 Decreases Liver Glucose Production and Increases Peripheral InsulinSensitivity

Hepatic glucose production and peripheral glucose utilization are bothcritical in the maintenance of normoglycemia and adaptation to feedingand fasting responses. Recent studies demonstrated that hepatic glucoseproduction and liver gluconeogenic activity were regulated by aP2 (Caoet al., (2013) Cell Metab. 17, 768-778), and suggested that thisresponse, alone or in combination with peripheral effects, may becritical in mediating the anti-diabetic effect of aP2 blockade. Todetermine whether this underlies the therapeutic properties of CA33,livers from HFD-fed mice after 5 weeks of CA33 or vehicle treatment werecollected. A marked reduction in expression of gluconeogenic genesphosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase(G6pc) in samples from the CA33-treated obese mice was observed (FIG.6A). In addition, the enzymatic activities of cytoplasmic PCK1 andmicrosomal glucose-6-phosphatase were significantly reduced in samplesfrom CA33-treated mice (FIG. 6B, C). These findings were consistent withearlier studies of aP2 function on liver (Cao et al., (2013) Cell Metab.17, 768-778).

Next whole body glucose fluxes by hyperinsulinemic-euglycemic clampstudies were examined (FIG. 6D). For these experiments mice were kept onHFD for 20 weeks prior to antibody treatment, and clamp studies wereperformed after 5 weeks of antibody intervention. During the clampstudy, CA33 treated obese mice required significantly higher glucoseinfusion rates to maintain euglycemia (FIG. 6E) and showed decreasedclamp hepatic glucose production (FIG. 6F), as well as a non-significanttrend towards a decrease basal hepatic glucose production (FIG. 8A,p=0.07). CA33 treatment also significantly increased whole body clampglucose disposal rates (RD) (FIG. 6G), although plasma insulin levelswere decreased compared to controls despite equivalent insulin infusion(5 mU/kg/min) (FIG. 8B). Taken together, these data indicate that CA33also increased whole body systemic insulin sensitivity and increasedinsulin-stimulated glucose utilization. To further assess glucoseutilization, we collected peripheral tissues after the clamp experimentand performed a 14C 2-deoxyglucose uptake assay. Insulin stimulatedglucose uptake was significantly higher in muscle tissue isolated fromCA33-treated obese mice (FIG. 4H), and there was a trend towards anincrease in glucose uptake in perigonadal white adipose tissue (PGWAT)(FIG. 8C, p=0.1). Furthermore, whole body glycolysis, measured as therate of increase in plasma [3H₂O] as a byproduct of glycolysis, was alsoincreased by CA33 treatment (FIG. 6I). These data support the conclusionthat the glucose-lowering effect of CA33 occurs predominantly throughdecreasing glucose production in liver and increasing glucoseutilization in peripheral tissues such as muscle.

Example 2 Anti-aP2 Monoclonal Antibody (CA33) for the Treatment of Type1 Diabetes CA33 Treatment is Protective Against Hyperglycemia andMortality in the NOD Mouse Model of Type 1 Diabetes

A model of Type 1 diabetes (T1D) in which diabetes onset is spontaneous,the NOD mouse (S. Makino, K. Kunimoto, Y. Murakoa, Y. Mizushima, K.Katagiri, Y. Tochino. Breeding of a non-obese, diabetic strain of mice.Jikken-dobutsu, Experimental animals 29, 1-13 (1980)), was used toexamine the effects of CA33 treatment on diabetes incidence andmortality. Animal care and experimental procedures were performed withapproval from animal care committees of Harvard University. Eight-weekold female NOD mice were injected subcutaneously with either vehicle orCA33. For the first 6 weeks of treatment the CA33 treated mice wereinjected with 1 mg/injection, which corresponds to ˜45 mg/kg dose, twiceweekly. Starting at the seventh week of the study CA33 treated mice wereinjected with a 30 mg/kg dose of CA33 twice weekly. Six hour fastingblood glucose was measured weekly. Incidence of diabetes was defined as2 consecutive weeks of 6 hour fasting blood glucose >200 mg/dL.

As seen in FIG. 9, treatment of NOD mice with anti-aP2 monoclonalantibody (CA33) was protective against the development of hyperglycemia.As a result, treatment with CA33 also reduced the mortality rate of NODmice in this experiment (FIG. 10A). Furthermore, treatment of NOD micewith anti-aP2 antibody from 10 weeks of age for 22 weeks (until 32 weeksof age) reduced fasting glycemia (6 hr fast) at 11, 12, and 15 weeks oftreatment (FIG. 10B) and reduced fasting insulin levels (6 hr fast) at16 weeks of treatment (FIG. 10C), as compared to vehicle-treated NODmice.

aP2 Deficiency Results in Improved Glucose Tolerance in NOD Mice Due toEnhanced Insulin Secretion and Expanded Beta-Cell Mass

It has previously been reported that blocking aP2 function via geneticdeletion, small molecule inhibition or antibody-mediated neutralizationis protective in mouse models of obesity-induced glucose intolerance(Hotamisligil et al, 2006; Uysal et al., 1996; Furuhashi et al., 2007;Cao et al., 2013; Burak et al., 2015). aP2 deletion was explored to seeif it would similarly confer improvements in glucose homeostasis in leanNOD mice. Indeed. NOD aP2⁴′ mice displayed significantly improvedglucose tolerance compared to aP2^(+/+) controls both at weaning (datanot shown) and at 7-8 weeks of age (FIG. 11A). Notably, this effect onglucose homeostasis was lost by the time mice reached 11 weeks of age,in line with the predicted onset of insulitis in this model.

In the setting of obesity, blocking aP2 is associated with improvedinsulin sensitivity. However, altered insulin sensitivity in lean NODaP2^(−/−) mice compared to controls was not observed (FIG. 11B). Thisraised the possibility that improved glucose tolerance in NOD aP2^(−/−)mice was related to a beta-cell intrinsic effect of aP2 deficiency. Toevaluate the effect of aP2 genetic deletion on beta cell function,islets were isolated from NOD aP2^(+/+) and NOD aP2^(−/−) mice at 4, 7,and 12 weeks of age. At baseline, genotype was not associated withdifferences in insulin secretion. However, upon stimulation with highglucose, aP2^(−/−) islets secreted significantly more insulin per cellthan aP2^(+/+) controls (FIG. 12A). Interestingly, this enhanced insulinsecretion began to decline with age (FIG. 12B and FIG. 12C), andsignificant insulin secretion differences in islets isolated from 19week old mice was not observed (data not shown). This is in line withthe finding of a transient improvement in glucose tolerance in young NODaP2^(−/−) mice, and suggests that before the onset of insulitis,enhanced beta cell responsiveness at least in part underlies the glucosehomeostasis phenotype in NOD aP2^(−/−) mice.

Improved glucose control observed in the setting of aP2 deficiency wasalso related to changes in beta cell mass. Remarkably, during pancreaticdissection more islets were visible in NOD aP2^(−/−) mice than inaP2^(+/+) controls (FIG. 13). This observation was further validated byquantifying beta-cell mass in NOD aP2^(−/−) mice and controls. In 7-weekold animals, aP2 deficiency was associated with a significant (˜35%)increase in beta-cell mass (FIGS. 14A and 14B).

Hormonal aP2 Amplifies Glucose-Stimulated Insulin Secretion

Given the recent discovery that aP2 is an actively secreted hormone andthe finding that antibody-mediated aP2 neutralization is protective inmodels of Type 1 Diabetes, the circulating form of aP2 was furtherexplored to try and understand whether this molecule acts directly onbeta cells to regulate their function, survival, or proliferation. Whileaddition of aP2 to primary islets under control, nutrient-abundantconditions had no effect on glucose-stimulated insulin secretion (GSIS)(FIG. 15A, FIG. 15B, and FIG. 15C), under starvation conditions (i.e.,conditions where FOXO1 is activated), addition of aP2 acts to potentiateGSIS (FIG. 15E and FIG. 15F). A Western blot was run to confirm that aP2is in fact taken up into mouse islets after 20 minutes of treatment with10 ug/ml aP2 (FIG. 15D). This potentiation of glucose-responsive insulinsecretion following prolonged starvation may be associated withincreased stress-resistance in response to nutrient depletion. Whilethis may be acutely beneficial, chronic activation of FOXO1 has beenshown to be deleterious to beta cell function, resulting in loss ofinsulin expression, impaired GSIS, beta cell dedifferentiation, andincreased susceptibility to apoptosis due to loss of PDX1 activity. Thusin the context of T1D, chronic activation of beta cell FOXO1 mayincrease susceptibility to immune destruction and accelerate diseasesdevelopment.

Monoclonal Antibody Targeting aP2 (CA33) is Effective in Reducing theIncidence of Diabetes in the RIP-LCMV-GP Mouse Model of Type 1 Diabetes

Neutralization of secreted aP2 provided beneficial effects in Type 1diabetes, also referred to as juvenile diabetes. Monoclonal antibodytargeting of aP2 with CA33 was effective in reducing the incidence ofdiabetes in an art-recognized rat insulin promoter-lymphocyticchoriomeningitis virus-glycoprotein (RIP-LCMV-GP) model of type 1diabetes.

The RIP-LCMV-GP mouse model of type 1 diabetes is characterized by highpenetrance and rapid onset of diabetes due to virally mediated β-celldestruction (FIG. 16) (Oldstone et al., (1991) Cell 65:319-331). TheRIP-LCMV-GP mice were a gift from K. Bornfeldt's laboratory atWashington University in Seattle. The genetic backgrounds of allintercrossed mouse models were verified by congenic genotyping (288 locifor C57BL/6) with an ABI 3130 analyzer. In the RIP-LCMV-GP model,diabetes was induced with the administration of LCMV (2×10³ PFU/ml).Animal care and experimental procedures were performed with approvalfrom animal care committees of Harvard University.

The experiment was performed on three groups of mice (FIG. 17). Group Aconsisted of 8 RIP-LCMV-GP (GP+) mice that were injectedintraperitoneally with vehicle (PBS) twice weekly starting at Day −14.Group B consisted of 8 RIP-LCMV-GP (GP+) mice that were injectedintraperitoneally with CA33 at a dose of 1.5 mg/injection twice weeklystarting at Day −14. Group C consisted of 8 RIP-LCMV-GP (GP+), aP2−/−mice that were injected intraperitoneally with vehicle twice weeklystarting at Day −14. At Day 0, diabetes was induced with theadministration of LCMV (2×10³ PFU/ml). Treatment with vehicle or CA33was continued for two weeks after administration of LCMV (FIG. 17).

Six-hour fasted blood glucose levels were measured periodically, and theonset of diabetes was defined as a 6-hour fasting blood glucose above250 mg/dL. GP+ vehicle-treated animals showed a significant rise infasting blood glucose levels (FIG. 18) and a greater than 75% incidenceof diabetes within 14 days from LCMV treatment (FIG. 19). GP+, aP2−/−mice showed overall, significantly lower blood glucose levels than GP+vehicle treated mice after LCMV treatment (FIG. 18). GP+, aP2−/− micealso experienced a much lower incidence of diabetes than the GP+ vehicletreated mice, 15% vs. 75% incidence (FIG. 19). Monoclonal antibodytreated GP+ mice showed significantly lower blood glucose levels thanthe vehicle treated control mice (FIG. 18) and incidence of diabeticphenotype was not observed in the monoclonal antibody treated group(FIG. 19).

These results demonstrate that reduction in aP2 levels either throughgenetic deficiency or by monoclonal antibody neutralization has a stronganti-diabetic effect in this virally induced model of Type 1 diabetes.

Monoclonal Antibody Targeting aP2 (CA33) is Effective in ReducingInflammation in the RIP-LCMV-GP Mouse Model of Type 1 Diabetes

The RIP-LCMV-GP mouse model of type 1 diabetes is described above.Inflammation was examined by determining insulitis levels in threegroups of mice. Group A consisted of 8 RIP-LCMV-GP (GP+) mice that wereinjected intraperitoneally with vehicle (PBS) twice weekly starting atDay −14. Group B consisted of 8 RIP-LCMV-GP (GP+) mice that wereinjected intraperitoneally with CA33 at a dose of 1.5 mg/injection twiceweekly starting at Day −14. Group C consisted of 8 RIP-LCMV-GP (GP+),aP2−/− mice that were injected intraperitoneally with vehicle twiceweekly starting at Day −14. At Day 0, diabetes was induced with theadministration of LCMV (2×10³ PFU/ml). Treatment with vehicle or CA33continued for two weeks after administration of LCMV.

Insulitis scoring was performed on hematoxylin and eosin (H&E) stainedpancreatic sections. Each islet is scored as either “non-insulitis,”“peri-insulitis,” “mild insulitis” or “severe insulitis.” The bar graphrepresents the percentage of each islet type in each animal analyzed(FIG. 20A).

For antibody staining, pancreata from RIP-LCMV-GP mice wereformalin-fixed and paraffin-embedded. Then, 5-μm serial sections of thepancreata were generated, and staining was performed with antibodiesagainst insulin (Linco), ATF6 (Santa Cruz Biotechnology); phospho-eIF2α(Biosource): sXBP1 (in house) and Alexa Fluor 488 and Alexa Fluor 568(Invitrogen) according to established protocols.

After staining, image analysis was performed by using custom softwaredeveloped in MATLAB (The MathWorks Inc.). Briefly, islet regions wereidentified as contiguous areas (connected pixels) of insulin staining(green channel) at or above a threshold intensity value optimized acrossmultiple images. Mean fluorescence intensity for insulin (green channel)and for either sXBP1 or ATF6 (red channel) was calculated as the sum ofintensities for all pixels divided by the number of pixels within theislet.

As shown in FIG. 20A, GP+ vehicle-treated animals showed high levelsinflammation with most islets showing either mild or severe insulitis.GP+, aP2−/− mice showed decreased levels of inflammation with mostislets showing mild insulitis (FIGS. 20A and 20D). Monoclonal antibody(CA33) treated GP+ mice also showed decreased levels of inflammationwith decreased numbers of islets showing severe insulitis (FIGS. 20A and20E).

As shown in the images in FIG. 21A, islet morphology and endoplasmicreticulum (ER) adaptive capacity was preserved in aP2 deficient andCA33-treated RIP-LCMV-GP mice, as shown by the increased levels of ATF6and sXBP1 staining. ATF6 levels were significantly increased in both theGP+, aP2−/− mice and the monoclonal antibody (CA33) treated GP+ mice(FIG. 21B). In addition, sXBP1 levels were also significantly increasedin both the GP+, aP2−/− mice and the monoclonal antibody (CA33) treatedGP+ mice (FIG. 21C).

These results demonstrate that reduction in aP2 activity levels eitherthrough genetic deficiency or by monoclonal antibody neutralizationreduces inflammation and preserves islet morphology in this virallyinduced model of Type 1 diabetes.

Example 3 Anti-aP2 Monoclonal Antibody for the Treatment, Reduction orPrevention of Atherosclerosis Animals

Animal care and experimental procedures were performed with approvalfrom animal care committees of Harvard University. Male ApoE−/− mice inthe C57BL/6J background (Jackson Laboratory) were kept on a 12-h lightcycle and were fed a high-cholesterol atherogenic western diet (D12079B:21% fat, 0.21% cholesterol; Research Diets) ad libitum, beginning at 4-5weeks of age. The mice were treated by subcutaneous administration ofvehicle or antibody (33 mg/kg) for 12 weeks starting at 6-weeks of age.

Assessment of Atherosclerotic Lesions

Mice were sacrificed and flushed with saline and then 10% neutralbuffered formalin solution by injection through the left ventricle. Theaorta was dissected from the proximal aorta to the iliac bifurcation,and the aortae were pinned out in an en face preparation. En facepinned-out aortas were stained with Sudan IV.

Quantitation of lesion areas was achieved using ImageJ softwaredeveloped at the NIH. The outer perimeter of the pinned out aorta wasdefined in the software to establish the total area of the aorta as awhite background. The percent area of the lesions stained red with SudanIV was then measured and calculated by the software.

As shown in FIG. 22B, ApoE knockout mice treated with CA33 had decreasedatherosclerotic lesion area. In contrast, ApoE knockout mice treatedwith CA15 showed no significant difference in atherosclerotic lesionarea compared to controls. Representative images of en face pinnedaortas from an ApoE knockout mice are shown in FIG. 22C (vehicle-treatedmice), FIG. 22D (CA33-treated mice), and FIG. 22E (CA15-treated mice).

The deletion efficiency of mice that aP2 is specifically knocked outwith the lox-P system in adipose tissue (aP2^(adip−/−) mice) wasanalyzed by western blot (FIG. 22G). aP2^(adip−/−) mice were crossed toApoE^(−/−) mice to generate ApoE^(−/−)aP2^(adip−/−) mice.ApoE^(−/−)aP2^(adip+/+) and ApoE^(−/−)aP2^(adip−/−) mice fed westerndiet for 12 weeks were analyzed for serum aP2, TG, and cholesterollevels (FIG. 22H, FIG. 22I, and FIG. 22J). As shown in FIG. 22I and FIG.22J, ApoE^(−/−)aP2^(adip−/−) mice had lower triglyceride and cholesterollevels compared to ApoE^(−/−)aP2^(adip+/+) mice. After sacrifice ofthese mice, atherosclerotic lesion area was analyzed by Sudan IVstaining of en face aorta from these mice (FIG. 22K and FIG. 22L). Asshown in FIG. 22K, ApoE^(−/−)aP2^(adip−/−) mice had decreasedatherosclerotic lesion area compared to ApoE^(−/−)aP2_(adip+/+) mice.Overall, these results show that adipose tissue derived aP2 regulatesdyslipidemia and atherosclerosis development in ApoE^(−/−) mice.

Body Weight and Liver Weight

ApoE knockout mice were fed on western diet beginning at 4-5 weeks ofage until mice were 18 weeks old. The mice were treated by subcutaneousadministration of vehicle or antibody (33 mg/kg) for 12 weeks startingat 6-weeks of age. Body weight, lean mass, and fat mass were measured bydual X-ray absorbance (DXA) spectroscopy.

As shown in FIG. 23A, ApoE knockout mice treated with CA33 antibodyshowed a significant decrease in body weight. In addition, ApoE knockoutmice treated with CA33 also showed a significant decrease in liverweight (FIG. 23B). In contrast, ApoE knockout mice treated with CA15 didnot show any significant differences in body weight or liver weight(FIGS. 23A, 23B, and 23C).

As shown in FIG. 23D, ApoE knockout mice treated with CA33 antibody didnot reveal any significant decreases in lean mass or fat mass. Inaddition, ApoE knockout mice treated with CA15 antibody did not show anysignificant differences in lean mass or fat mass (FIG. 23D).

Glucose Tolerance Test

The glucose tolerance test was performed by oral glucose administration(1.0 g/kg) on conscious mice after an overnight (16 h) fast. Particlesize distribution of the lipoproteins was determined by fast-performanceliquid chromatography (FPLC), using pooled samples of plasma after 6 or12 weeks of vehicle or antibody treatment (33 mg/kg).

As shown in FIG. 24A, CA33 treated mice have a statistically significantlower fasting blood glucose than vehicle treated mice. However, CA33treated mice did not show any difference in response to glucose in theglucose tolerance test (GTT) (FIG. 24B).

Measurement of Cholesterol and Triglycerides in Lipoprotein Fractions

Cholesterol and triglycerides were measured in lipoprotein fractions(total lipoprotein, very low-density lipoprotein (VLDL), low-densitylipoprotein (LDL), or high-density lipoprotein (HDL)) in ApoE knockoutmice treated with PBS, CA33 (33 mg/kg), or CA15 (33 mg/kg) for six weeksor twelve weeks. Particle size distribution of the lipoproteins wasdetermined by fast-performance liquid chromatography (FPLC), usingpooled samples of plasma.

As shown in FIG. 25A, ApoE knockout mice treated with CA33 for six weeksshowed significant decreases in cholesterol levels in total lipoprotein,very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), andhigh-density lipoprotein (HDL). In addition, ApoE knockout mice treatedwith CA33 for twelve weeks showed significant decreases in cholesterollevels in total lipoprotein, very low-density lipoprotein (VLDL),low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (FIG.25B). Treatment of ApoE knockout mice with CA15 for six weeks did notyield any significant differences in cholesterol levels (FIG. 25A),while treatment of ApoE knockout mice with CA15 for twelve weeks led tosmaller decreases in cholesterol in total lipoprotein, low-densitylipoprotein (LDL), and high-density lipoprotein (HDL), when compared toCA33-treated animals (FIG. 25B).

As shown in FIG. 26A, ApoE knockout mice treated with CA33 for six weeksshowed significant decreases in triglyceride levels in total lipoproteinand very low-density lipoprotein (VLDL). In addition, ApoE knockout micetreated with CA33 for twelve weeks showed significant decreases intriglyceride levels in total lipoprotein, very low-density lipoprotein(VLDL), low-density lipoprotein (LDL), and high-density lipoprotein(HDL) (FIG. 26B). In contrast, treatment of ApoE knockout mice with CA15for six weeks or twelve weeks did not yield any significant differencesin triglyceride levels (FIGS. 26A and 26B), as compared to controlanimals treated with PBS.

Example 4 Humanization of CA33

Rabbit Antibody 909 (CA33) was humanised by grafting the CDRs from therabbit CDR/mouse framework hybrid antibody V-region CDRs onto humangermline antibody V-region frameworks. In order to recover the activityof the antibody, a number of framework residues from the rabbit/mousehybrid V-region were also retained in the humanized sequence. Theseresidues were selected using the protocol outlined by Adair et al.(1991) (Humanised antibodies. WO91/09967). Alignments of therabbit/mouse hybrid antibody (donor) V-region sequences with the humangermline (acceptor) V-region sequences are shown in FIG. 27 (VL) andFIG. 28 (VH), together with the designed humanized sequences. The CDRsgrafted from the donor to the acceptor sequence are as defined by Kabat(Kabat et al., 1987), with the exception of CDRH1 where the combinedChothia/Kabat definition is used (see Adair et al., 1991 Humanisedantibodies. WO91/09967).

Genes encoding a number of variant heavy and light chain V-regionsequences were designed and constructed by an automated synthesisapproach by DNA 2.0 Inc. Further variants of both heavy and light chainV-regions were created by modifying the VH and VK genes byoligonucleotide-directed mutagenesis, including, in some cases,mutations within CDRs to modify potential aspartic acid isomerisationsites or remove unpaired Cysteine residues. These genes were cloned intovectors to enable expression of humanized 909 IgG4P (human IgG4containing the hinge stabilising mutation S241P, Angal et al., MolImmunol. 1993, 30(1):105-8) antibodies in mammalian cells. The varianthumanized antibody chains, and combinations thereof, were expressed andassessed for their potency relative to the parent antibody, theirbiophysical properties and suitability for downstream processing,leading to the selection of heavy and light chain grafts shown in FIGS.24 and 25.

Human V-region IGKV1-17 (A30) plus JK4 J-region was chosen as theacceptor for antibody 909 light chain CDRs. The light chain frameworkresidues in grafts gL1 (Seq. ID No. 446), gL10 (Seq. ID No. 448), gL54(Seq. ID No. 450) and gL55 (Seq. ID No. 452) are all from the humangermline gene, with the exception of residues 2, 3, 63 and 70 (Kabatnumbering), where the donor residues Valine (2V), Valine (3V), Lysine(63K) and Aspartic acid (70D) were retained, respectively. Retention ofresidues 2, 3, 63 and 70 was essential for full potency of the humanizedantibody. Residue 90 in CDRL3 of the gL10 graft, gL54 graft, and gL55graft was mutated from a Cysteine (90C) to a Serine (90S), Glutamine(90Q), and Histidine (H90) residue, respectively, thus removing theunpaired Cysteine residue from the gL10, gL54, and gL55 sequence.

Human V-region IGHV4-4 plus JH4 J-region was chosen as the acceptor forthe heavy chain CDRs of antibody 909. In common with many rabbitantibodies, the VH gene of antibody 909 is shorter than the selectedhuman acceptor. When aligned with the human acceptor sequence, framework1 of the VH region from antibody 909 (Seq. ID No. 454) lacks theN-terminal residue, which is retained in the humanised antibody (FIG.28). Framework 3 of the 909 rabbit VH region also lacks two residues (75and 76) in the loop between beta sheet strands D and E: in graft gH1(Seq. ID No. 455) the gap in framework 3 is conserved, whilst in graftgH4 (Seq. ID No. 457), gH15 (Seq. ID No. 459), gH61 (Seq. ID No. 461),and gH62 (Seq. ID No. 463) the gap is filled with the correspondingresidues (Lysine 75, 75K; Asparagine 76, 76N) from the selected humanacceptor sequence (FIG. 28). The heavy chain framework residues ingrafts gH1 and gH15 are all from the human germline gene, with theexception of residues 23, 67, 71, 72, 73, 74, 77, 78, 79, 89 and 91(Kabat numbering), where the donor residues Threonine (23T),Phenylalanine (67F), Lysine (71K), Alanine (72A), Serine (73S),Threonine (74T), Threonine (77T), Valine (78V), Aspartic acid (79D),Threonine (89T) and Phenylalanine (91F) were retained, respectively. Theheavy chain framework residues in graft gH14 are from the human germlinegene, with the exception of residues 67, 71, 72, 73 74, 77, 78, 79, 89and 91 (Kabat numbering), where the donor residues Threonine (23T),Phenylalanine (67F), Lysine (71K), Alanine (72A), Serine (73S),Threonine (74T), Threonine (77T), Valine (78V), Aspartic acid (79D),Threonine (89T) and Phenylalanine (91F) were retained, respectively. Theheavy chain framework residues in grafts gH61 and gH62 are from thehuman germline gene, with the exception of residues 71, 73, and 78(Kabat numbering), where the donor residues Lysine (71K), Serine (73S),and Valine (78V) were retained, respectively. The Glutamine residue atposition 1 of the human framework was replaced with Glutamic acid (1E)to afford the expression and purification of a homogeneous product: theconversion of Glutamine to pyroGlutamate at the N-terminus of antibodiesand antibody fragments is widely reported. Residue 59 in CDRH2 (Seq. IDNo. 19) of the gH15 graft and gH62 graft was mutated from a Cysteine(59C) to a Serine (59S) residue, thus removing the unpaired Cysteineresidue from the gH15 sequence. Residue 98 in CDRH3 (Seq. ID No. 20) ofgraft gH15 and graft gH62 was mutated from an Aspartic acid (98D) to aGlutamic acid (98E) residue, thus removing a potential Aspartic acidisomerization site from the gH15 sequence.

For expression of humanized Ab 909 in mammalian cells, the humanizedlight chain V-region gene was joined to a DNA sequence encoding thehuman C-kappa constant region (K1m3 allotype), to create a contiguouslight chain gene. The humanized heavy chain V-region gene was joined toa DNA sequence encoding the human gamma-4 heavy chain constant regionwith the hinge stabilising mutation S241P (Angal et al., Mol Immunol.1993, 30(1):105-8), to create a contiguous heavy chain gene. The heavyand light chain genes were cloned into the mammalian expression vector1235-pGL3a(1)-SRHa(3)-SRLa(3)-DHFR(3) (Cellca GmbH).

Example 5 Reducing Affinity of a High Affinity Anti-aP2 MonoclonalAntibody

In light of the therapeutic activity of the low-affinity anti-aP2monoclonal antibody CA33, amino acid residue substitutions were exploredin the high-affinity anti-aP2 monoclonal murine antibody (H3 gL1/gH1) inorder to develop low-affinity antibodies having potential aP2 modulatingtherapeutic activity. The original amino acid and cDNA sequences of H3gL1/gH1, along with the mutated sequences thereof, are provided in Table13 below.

TABLE 13 Modification of High Affinity H3 anti-aP2-antibody Seq.Protein/cDNA ID No. Sequence H3 gL1 light 485EVVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYG chain variableTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSHYPLTFGAG region TKLELK H3 gL1light 502 GAAGTTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGG chainvariable AGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAA region (Seq.CTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATT ID No. 485)TATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAG cDNATGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTCATTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA H3 gL11 light 486EVVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYG chain variableTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQASHYPLTFGAG region TKLELK H3gL11 light 503 GAAGTTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGG chainvariable AGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAA region (Seq.CTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATT ID No. 486)TATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAG cDNATGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGGCGAGTCATTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA H3 gH1 heavy 482QVQLQQPGAELVKPGASVKLSCKASGYTFTSNWITWVKQRPGQGLEWIGD chain variableIYPGSGSTTNNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARLRGY regionYDYFDFWGQGTTLTVSS H3 gH1 heavy 504CAGGTCCAACTACAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGG chain variableCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC region (Seq.AACTGGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGT ID No. 482)GGATTGGAGATATTTATCCTGGTAGTGGTAGTACTACTAACAATGAG cDNAAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTAAGGGGCTACTATGATTACTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCGAGT H3 gH19 483QVQLQQPGAELVKPGASVKLSCKASGYTFTSNAITWVKQRPGQGLEWIGD heavy chainISPGSGSTTNNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARLRGY variable regionYDYFDFWGQGTTLTVSS H3 gH19 505CAGGTCCAACTACAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGG heavy chainCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC variable regionAACGCGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGT (Seq. ID No.GGATTGGAGATATTTCTCCTGGTAGTGGTAGTACTACTAACAATGAG 483) cDNAAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTAAGGGGCTACTATGATTACTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCGAGT H3 gH20 484QVQLQQPGAELVKPGASVKLSCKASGYTFTSNAITWVKQRPGQGLEWIGD heavy chainISPGSGSTTNNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARLRGFY variable regionDYFDFWGQGTTLTVSS H3 gH20 495CAGGTCCAACTACAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGG heavy chainCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC variable regionAACGCGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGT (Seq. ID No.GGATTGGAGATATTTCTCCTGGTAGTGGTAGTACTACTAACAATGAG 484) cDNAAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTAAGGGGCTTCTATGATTACTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCGAGT Heavy chain 496AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSG Fab gamma 1VHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC constant regionHeavy chain 497 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCC Fabgamma 1 CAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCT constantGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCAC region (Seq.ACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTGAGCAGCTCAGTGA ID No. 496)CTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCA cDNACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT Heavy chain 498AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT full lengthFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK gamma 1PCICTVPEVSSVFIFPPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV constantEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT regionISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK Heavy chain499 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCC full lengthCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCT gamma 1GAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCAC constantACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGA region (Seq.CTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCA ID No. 498)CCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGG cDNATTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGATCCCAGTGTCCTTGGAGCCCTCTGGTCCTACAGGACTCTGACACCTACCTCCACCCCTCCCTGTATAAA Light chain 500RTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL kappa constantNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Light chain 501CGTACGGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT kappa constantAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAG (Seq. ID No.ACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCT 500) cDNAGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT

To reduce the affinity of H3 (gL1/gH1) to that of CA33, H3 residueslikely to form key contacts with murine aP2 were identified by visualinspection of the crystal structure (FIG. 29), and mutated to Alanine,Phenylalanine, or Serine. Initially, a panel of 11 point mutationsacross 9 positions were generated and ranked using SPR analysis.Mutations that showed significant reductions in affinity were combinedand then ranked using the Octet. Further combinations were produceduntil the affinity of CA33 was roughly matched.

By substituting the amino acid residue W91 in the light chain parentCDRL3 region to Ala, W33 in the heavy chain CDRH1 region to Ala, and Y52in the heavy chain CDRH2 region to Ser (H3 gL11/gH19), affinity wasreduced from <0.07 μM. By substituting the amino acid residue W33 in theheavy chain CDRH1 region to Ala, Y52 in the heavy chain CDRH2 region toSer, and Y98 in the heavy chain CDRH3 region to Phe (H3 gL1/gH20),affinity was reduced from <0.07 μM. The full variable chains of theresultant antibodies and their alignment are provided in FIGS. 30B & C.

Additional experiments examining the binding affinities of anti-aP2antibodies that have been mutated to reduce binding affinity(5251.mIgG1168 (PB1172) and 5252.mIgG1.169 (PB1171)) are shown in FIG.31. PB1172 is comprised on the g11 light chain and g19 heavy chain (SeeFIGS. 30B & C) and PB1171 is comprised on the g1 light chain and g20heavy chain (See FIGS. 30B & C). Binding affinities toward human aP2were 6.03 M and 5.82 M for the PB1172 and PB1171 antibodies,respectively. Binding affinities toward mouse aP2 were 4.57 μM and 5.12μM for the PB1172 and PB1171 antibodies, respectively.

Example 6 Adipokine aP2 Regulates Hepatic Glucose Production byDisrupting a Repressive FoxO1/CtBP2 Transcriptional Complex Animals

Leptin-deficient ob/ob mice (10 weeks of age) and high-fat diet-inducedobese mice (12-16 weeks on high-fat diet) were purchased from theJackson Laboratories (Bar Harbor, Me.). All mice used were males andmaintained on a 12 h dark/light cycle. All experimental proceduresinvolving animals were conducted according to the guidelines of theInstitutional Animal Care and Use Committee, Harvard School of PublicHealth.

1) aP2 Knockout Mice on High-Fat Diet Study.

Wild-type or aP2-deficient mice [see, Hotamisligil, G. S., et al.Uncoupling of obesity from insulin resistance through a targetedmutation in aP2, the adipocyte fatty acid binding protein. Science 274,1377-1379 (1996)] on C57BL6/J background were fed either a high-fat diet(60 kcal %, Research Diets, Inc., NJ, D12492i) or normal chow diet (RD,PicoLab 5058 Lab Diet) from 8 weeks of age for 16 weeks.

2) Treatment of Diet-Induced Obese Mice with aP2 Antibody.

Wild-type mice were fed a high-fat diet for 12 weeks to induce dietaryobesity and thereafter treated with either vehicle or monoclonal aP2antibody (1.5 mg CA33/mouse) twice a week for 4 weeks on the same diet.

3) Recombinant aP2 Protein Injection Study.

Wild-type mice (10-12 weeks of age) received intraperitoneal injectionsof either vehicle or recombinant aP2 protein (100 μg/mouse) twice dailyfor 5 days. FoxO1^(flox/flox) mice were generously provided by DomenicoAccili (Columbia University, NY) and crossed with Albumin-Cre mice(Jackson Laboratories, Bar Harbor, Me.) to generate liver specific FoxO1knockout mice. Either wild-type mice or FoxO1 liver specific knockoutmice were treated with recombinant aP2 in the same way.

4) Adenovirus Transduction Study.

Recombinant adenovirus encoding either β-glucuronidase (AdGUS), CtBP2(AdCtBP2), sh-control (Ad/shControl), sh-FoxO1 (Ad/shFoxO1) or sh-CtBP2(Ad/shCtBP2) was delivered into mice at the titer of 1.5×10¹¹particles/mouse. Glucose tolerance tests were performed byintraperitoneal glucose injection (0.75 g/kg) after an overnight fast,and insulin tolerance tests were performed by intraperitoneal injectionof insulin (0.5 U/kg) after a 6 h-fast. Serum parameters were analyzedusing the following systems: aP2; Mouse FABP4 ELISA (BioVendor),insulin; Mouse Ultrasensitive Insulin ELISA (ALPCO), glucagon; GlucagonQuantikine ELISA Kit (R&D Systems), FFA; NEFA-HR(2) (Wako Chemicals),glycerol; Free Glycerol Determination Kit (Sigma), Alanineaminotransferase (ALT); ALT activity assay kit (Sigma). Livertriglyceride was extracted by the chloroform-methanol method anddetermined by a colorimetric assay (Sigma).

Recombinant Protein Production and Purification

Mouse aP2 cDNA with an N-terminal polyhistidine tag followed by a TEVprotease cleavage site was cloned into pET28 vector (EMD bioscience) andthe plasmid was transformed into BL21AI strain of E. coli (Invitrogen).After the affinity purification and extensive endotoxin removal, theN-terminal His tag was removed by cleavage with TEV protease (Sigma).Lipid binding mutant (R126L, Y128F) was generated following the samestrategy [see, Erbay, E., et al. Reducing endoplasmic reticulum stressthrough a macrophage lipid chaperone alleviates atherosclerosis. NatureMedicine 15, 1383-1391 (2009)]. To delipidate the recombinant protein,temperature-dependent binding of hydrophobic ligands to Lipidex1000(Perkin-Elmer, Norwalk, Conn.), a 10% substituted hydroxyalkoxypropylderivate of Sephadex G-25, was utilized. At 37° C., Lipidex 1000 removesboth non-protein bound and protein associated lipids from an aqueoussolution, whereas at 0° C. it only removes non-protein bound lipids[see, Glatz, J. F. & Veerkamp, J. H. Removal of fatty acids from serumalbumin by Lipidex 1000 chromatography. Journal of Biochemical andBiophysical Methods 8, 57-61 (1983)]. Recombinant aP2 protein wasdelipidated with Lipidex1000 at 37° C. twice. Thereafter, the resultantproteins were further purified by gel filtration through Sephadex G-25(PD-10, GE Healthcare).

Primary Hepatocyte Isolation, Screening of Transcription Factors,Subcellular Fractionation, and Adenovirus Transduction

Primary mouse hepatocytes were isolated as described previously, see,Sekiya, M., et al. SREBP-1-independent regulation of lipogenic geneexpression in adipocytes. Journal of Lipid Research 48, 1581-1591(2007). After overnight serum starvation, cells were treated inWilliam's medium E (Invitrogen) with 50 μg/ml of recombinant aP2 in thepresence or absence of forskolin (2 μM, Sigma) for 3 h unless otherwiseindicated. For the screening of transcription factors, on-target plussiRNAs were purchased from Dharmacon and transiently transfected intoprimary hepatocytes by RNAiFect (QIAGEN). For subcellular fractionation,cells were treated as indicated above for 90 min and fractionatedutilizing NE-PER cell fractionation system (Pierce). For adenoviraltransduction, shRNA oligonucleotides were cloned into pENTR/U6 vectorfollowed by the recombination with pAd/BLOCK-it-DEST vector(Invitrogen). The targeted sequences of shRNAs were designed as follows.Control; 5′-GTCTCCACGCGCAGTACATTT-3′, Seq. ID No. 541 Foxo1;5′-GCATGTTTATTGAGCGCTTGG-3′, Seq. ID No. 542 CtBP1;5′-GCAGCGGGTTTGACAATATCG-3′, Seq ID No. 543 CtBP2;5′-GGGAAGACTAGGACGTGATTA-3′ Seq. ID No. 544 and5′-GCCACATTCTCAATCTGTATC-3′, Seq. ID No. 545 HNF4a;5′-GCTGCAGATTGATGACAATGA-3′ Seq. ID No. 546. Forkhead response element(FHRE) luciferase (Addgene, Cambridge, Mass.) was cloned intopAd/PL-DEST vector (Invitrogen). Adenovirus encoding renilla luciferasewas purchased from Vector Biolabs (Philadelphia, Pa.). Adenoviruses wereamplified in HEK293A cells and purified by CsCl gradient centrifugation.To measure FoxO transcriptional activity, primary hepatocytes wereinfected with adenovirus expressing FHRE luciferase and renillaluciferase for 24 h. Thereafter, cells were serum starved overnight andincubated with 50 μg/ml of aP2 protein in the absence or presence of 2μM forskolin for 6 h. Cells were lysed in passive lysis buffer andanalyzed using Dual-Glo luciferase Reporter Systems (Promega). Fireflyluciferase signal was normalized to Renilla luciferase.

Quantitative Real-Time RT-PCR

Total RNA was isolated using Trizol Reagent (Invitrogen) and cDNA wassynthesized with iScript Reverse Transcription Supermix (Bio-Rad).Quantitative real-time PCR analysis was performed using SYBR Green inViiA 7 Real-Time PCR systems (Applied Biosystems). Data were normalizedto acidic ribosomal phosphoprotein P0 (Rplp0, 36B4) expression. Primersused for Q-PCR were as follows:

Rplp0 forward 5′-CACTGGTCTAGGACCCGAGAA-3′ Seq. ID No. 547 reverse5′-AGGGGGAGATGTTCAGCATGT-3′ Seq. ID No. 548 Pck1 forward5′-CTGCATAACGGTCTGGACTTC-3′ Seq. ID No. 549 reverse5′-CAGCAACTGCCCGTACTCC-3′ Seq. ID No. 550 G6pc forward5′-CGACTCGCTATCTCCAAGTGA-3′ Seq. ID No. 551 reverse5′-GTTGAACCAGTCTCCGACCA-3′ Seq. ID No. 552 Foxo1 forward5′-AACACACAGCTGGGTGTCAGG-3′ Seq. ID No. 553 reverse5′-GCATCTTTGGACTGCTCCTCAGT-3′ Seq. ID No. 554 Foxo3 forward5′-CTGGTGCTAAGCAGGCCTCAT-3′ Seq. ID No. 555 reverse5′-TGTAGGTCTTCCGTCAGTTTGAGG-3′ Seq. ID No. 556 Foxo4 forward5′-TGTATATGGAGAACCTGGAGTGCG-3′ Seq. ID No. 557 reverse5′-CAAAGCTTCTTGCTGTGACTCAGG-3′ Seq. ID No. 558 Cebpa forward5′-CAAGAACAGCAACGAGTACCGG-3′ Seq. ID No. 559 reverse5′-TGTCACTGGTCAACTCCAGCAC-3′ Seq. ID No. 560 Nr3c1 forward5′-CTGACGTGTGGAAGCTGTAAAGTC-3′ Seq. ID No. 561 reverse5′-GATGCAATCATTTCTTCCAGCAC-3′ Seq. ID No. 562 CtBP1 forward5′-TTGGGCATCATTGGACTAGGTC-3′ Seq. ID No. 563 reverse5′-GCTCGATTCCATCAGATAGGTATGG-3′ Seq. ID No. 564 CtBP2 forward5′-GCAGGACTTGCTATATCAGAGCGA-3′ Seq. ID No. 565 reverse5′-ATGCACCTTGCCTCATCTGCT-3′ Seq. ID No. 566 Alb forward5′-AGACGTGTGTTGCCGATGAGT-3′ Seq. ID No. 567 reverse5′-GTTTTCACGGAGGTTTGGAATG-3′ Seq. ID No. 568 Gpam forward5′-ACAGTTGGCACAATAGACGTTT-3′ Seq. ID No. 569 reverse5′-CCTTCCATTTCAGTGTTGCAGA-3′ Seq. ID No. 570 Fasn forward5′-AGAGATCCCGAGACGCTTCT-3′ Seq. ID No. 571 reverse5′-GCCTGGTAGGCATTCTGTAGT-3′ Seq. ID No. 572 Scd1 forward5′-CCCGGGAGAATATCCTGGTTT-3′ Seq. ID No. 573 reverse5′-TCGATGAAGAACGTGGTGAAGT-3′ Seq. ID No. 574 Hif1a forward5′-ATGAAGTGCACCCTAACAAGCC-3′ Seq. ID No. 575 reverse5′-CACACTGAGGTTGGTTACTGTTGG-3′ Seq. ID No. 576 Ppargc1a forward5′-GAAGTGGTGTAGCGACCAATC-3′ Seq. ID No. 577 reverse5′-AATGAGGGCAATCCGTCTTCA-3′ Seq. ID No. 578 Stat3 forward5′-ACCATTGACCTGCCGATGTC-3′ Seq. ID No. 579 reverse5′-TGAGCGACTCAAACTGCCCT-3′ Seq. ID No. 580 Srebf1c forward5′-GGAGCCATGGATTGCACATT-3′ Seq. ID No. 581 reverse5′-GGCCCGGGAAGTCACTGT-3′ Seq. ID No. 582 Mlxip1 forward5′-CACTCAGGGAATACACGCCTAC-3′ Seq. ID No. 583 reverse5′-ATCTTGGTCTTAGGGTCTTCAGG-3′ Seq. ID No. 584 Pklr forward5′-TCAAGGCAGGGATGAACATTG-3′ Seq. ID No. 585 reverse5′-CACGGGTCTGTAGCTGAGTGG-3′ Seq. ID No. 586 Acly forward5′-ACCCTTTCACTGGGGATCACA-3′ Seq. ID No. 587 reverse5′-GACAGGGATCAGGATTTCCTTG-3′ Seq. ID No. 588 Crp forward5′-ATGGAGAAGCTACTCTGGTGCC-3′ Seq. ID No. 589 reverse5′-ACACACAGTAAAGGTGTTCAGTGGC-3′ Seq. ID No. 590 Apcs forward5′-GTCAGACAGACCTCAAGAGGAAAGT-3′ Seq. ID No. 591 reverse5′-AGGTTCGGAAACACAGTGTAAAATT-3′ Seq. ID No. 592 CD36 forward5′-AGATGACGTGGCAAAGAACAG-3′ Seq. ID No. 593 reverse5′-CCTTGGCTAGATAACGAACTCTG-3′ Seq. ID No. 594 Acadm forward5′-AGGGTTTAGTTTTGAGTTGACGG-3′ Seq. ID No. 595 reverse5′-CCCCGCTTTTGTCATATTCCG-3′ Seq. ID No. 596

Nuclear Fatty Acyl-CoA Content

Primary hepatocytes (6×10⁶ cells per sample) were stimulated with 50μg/ml of aP2 for 2 h. The cells were washed with PBS and nuclei wereisolated without detergent as described previously in T. Nakamura etal., A critical role for PKR complexes with TRBP in Immunometabolicregulation and eIF2alpha phosphorylation in obesity. Cell Rep 11,295-307 (2015). The lipids in the isolated nuclei were extractedbasically by Bligh-Dyer method using water acidified with acetic acid.Fatty acyl-CoA partitioned into methanol/water phase was collected.Fatty acyl-CoAs were oxidized by fatty acyl-CoA oxidase (Wako, Japan),yielding 2,3-trans-Enoyl-CoAs and hydrogen peroxide. The hydrogenperoxide was quantified by a fluorescent detection system (Enzo LifeSciences, NY).

Glucose Production Assay

Primary hepatocytes were infected with either Ad/shControl or Ad/shFoxO1for 24 h and further incubated in fresh William's E medium with 5% FBSfor 12 h. Thereafter, cells were serum starved overnight and incubatedwith either control or recombinant aP2 (50 μg/ml) in the presence orabsence of forskolin (2 μM) for 5 h. Cells were washed twice with DMEMwithout glucose (Sigma) supplemented with 10 mM HEPES and incubated withthe same DMEM without glucose with 20 mM glycerol in the presence of thesame concentrations of vehicle, aP2 and/or forskolin for 2 h. Theglucose concentrations in the media were determined by using the AmplexRed Glucose/Glucose Oxidase Assay Kit (Invitrogen).

Western Blot Analysis and Co-Immunoprecipitation Experiments

Proteins were extracted from cells or liver samples with buffer A (50 mMTris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM NaF, 2mM Na₃VO₄) with complete protease inhibitors (Sigma-Aldrich) andsubjected to SDS-polyacrylamide gel electrophoresis. To detectacetylated FoxO1, 5 μM of trichostatin A (TSA) and 5 mM of nicotinamidewere included in the buffer A to block deacetylation of FoxO1 protein.Membranes were incubated with anti-FoxO1 (Cell Signaling, C29H4),anti-CtBP (Santa Cruz, E-12), anti-CtBP1 (BD), anti-CtBP2 (BD),anti-FLAG (Clontech), anti-GAPDH (Santa Cruz, FL-335), anti-Lamin A/C(Cell Signaling, 4C11), anti-phospho PKA substrate (Cell signaling,#9621), anti-GUS (Invitrogen), anti-pAkt (Ser473, Cell Signaling,#9271), anti-Akt (Santa Cruz, H-136), anti-Ac-FKHR (Santa Cruz, D-19),anti-pFoxO1 (Ser 256, Cell Signaling, #9461) and anti-HNF4a (Santa Cruz,C-19). The membranes were incubated with the secondary antibodyconjugated with horseradish peroxidase (Santa Cruz) and were visualizedusing the enhanced chemiluminescence system (Roche Diagnostics). Todetect endogenous binding of FoxO1 and CtBPs, the anti-FoxO1 antibody(Santa Cruz, C-9), anti-CtBP antibody (Santa Cruz, E-12), anti-CtBP2(Santa Cruz, E-16), control mouse IgG (generated in house) or controlgoat IgG (Santa Cruz) were cross-linked to Protein G dynabeads(Invitrogen) with 50 mM dimethyl pimelimidate (Sigma-Aldrich). Primaryhepatocytes or liver samples were lysed with buffer A and the proteincomplex was immunoprecipitated in buffer A with reduced concentration ofNP40 (0.5%) (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mMEDTA, 10 mM NaF, 2 mM Na₃VO₄) for 4 h at 4° C. The beads were washedwith the buffer A with 0.5% NP40 without EDTA four times, eluted withSDS loading buffer and analyzed by Western blot analysis. FLAG-tagco-immunoprecipitation study was performed as follows. The plasmidsencoding FLAG wild-type FoxO1 and CtBP1 are generous gifts from DomenicoAccili (Columbia University, NY) and Pere Puigserver (Harvard MedicalSchool, MA), respectively. CtBP2 cDNA was amplified by PCR and clonedinto pcDNA3.1(+) (Invitrogen). The PSDL motif in FoxO1 was eithermutated to PSAS or deleted by gene tailor site-directed mutagenesissystem (Invitrogen). HEK293 cells were transiently transfected withcontrol plasmid, FLAG wild-type FoxO1 or mutated FLAG FoxO1 along witheither CtBP1 or CtBP2 using lipofectamine LTX (Invitrogen). Cells werelysed with buffer A with 1% NP40 and immunoprecipitated with FLAG M2magnetic beads (Sigma) in buffer A with 0.5% NP40 for 4 h at 4° C. Thebeads were washed four times with buffer A, with 0.5% NP40 and elutedwith 0.5 mg/ml of 3× FLAG peptide (Sigma). To evaluate the effect ofoleoyl-CoA and NADH, the cell lysates were immunoprecipitated with FLAGM2 magnetic beads with increasing concentrations of oleoyl-CoA or NADHfor 4 h at 4° C. Thereafter, the FoxO1/CtBP2 complex was eluted andanalyzed in the same way.

Fatty Acid Uptake Assay

[1-¹⁴C]oleate (59.0 mCi/mmol, PerkinElmer) was conjugated with BSA(fatty acid free, Sigma, BSA:oleate=1:6). Primary hepatocytes wereincubated with recombinant aP2 (50 μg/ml) for 2 h and [1-¹⁴C]oleate-BSAcomplex was added to give a final concentration of 150 μM. After theindicated period of time, cells were placed on ice, washed with cold PBSfive times and lysed with 0.1N NaOH to measure the radioactivity andprotein concentrations.

Fatty Acid Oxidation

Primary hepatocytes were seeded on type I collagen (Sigma)-coated XF96cell culture microplates (Seahorse Bioscience) at a density of7×10³/well. Cells were treated with recombinant aP2 (50 μg/ml) for 2 hin Williams' E media without serum, thereafter the culture medium waschanged to Krebs-Henseleit buffer (111 mM NaCl, 4.7 mM KCl, 1.25 mMCaCl₂, 2 mM MgSO₄, 1.2 mM NaH₂PO₄, 2.5 mM glucose, 0.5 mM carnitine, 5mM HEPES pH 7.4) with the same concentrations of vehicle or recombinantaP2. Oxygen (O₂) consumption rates (OCR) were measured before and afterthe exposure to 150 μM of palmitate conjugated with BSA (SeahorseBioscience).

Immunocytochemistry

Primary hepatocytes were incubated as described above for 90 min andfixed in 4% paraformaldehyde for 10 min at room temperature. Cells werepermeabilized with 0.1% Triton X-100 for 10 min and blocked with 10%donkey serum (Sigma) for 1 hour. Primary antibodies employed to detectCtBP2 and FoxO1 were goat polyclonal CtBP2 antibody E-16 (Santa Cruz,1:50) and a mixture of rabbit polyclonal FoxO1 antibodies containingab39670 (Abcam®, 1:100), C29H4 (Cell Signaling, 1:100) and H-128 (SantaCruz, 1:100), respectively. Alexa Fluor® 568-conjugated donkey anti-goatIgG and Alexa Fluor® 647-conjugated donkey anti-rabbit IgG were appliedas secondary antibodies (Life Technologies, 1:200).

Chromatin Immunoprecipitation (ChIP)

Primary hepatocytes were incubated as described above for 90 min andfixed in 1% formaldehyde for 10 minutes at room temperature.Crosslinking was quenched by adding glycine to a final concentration of125 mM. Thereafter, the ChIP assay was carried out using Magna ChIPHiSens Chromatin Immunoprecipitation Kit (EMD Millipore) with minormodifications: chromatin shearing was achieved by Micrococcal Nuclease(Cell Signaling). Chromatin was immunoprecipitated either with controlIgG, anti-FoxO1 (Abcam®, ab39670) or anti-CtBP2 (Santa Cruz, E-16).Immunoprecipitated DNA and input DNA were quantified by real-time PCRwith primers specific for G6pc gene promoter. See, Hall, J. A., Tabata,M., Rodgers, J. T. & Puigserver, P. USP7 attenuates hepaticgluconeogenesis through modulation of FoxO1 gene promoter occupancy.Molecular endocrinology 28, 912-924 (2014).

Cellular Lactate/Pyruvate Measurement

To estimate the cytosolic NAD⁺/NADH ratio, cellular lactate and pyruvatecontents were measured with a kit (Cayman Chemical). When the conversionbetween pyruvate/NADH and lactate/NAD is at equilibrium, the cytosolicNAD⁺/NADH ratio could be estimated by the lactate/pyruvate ratio. See,Williamson, D. H., Lund, P. & Krebs, H. A. The redox state of freenicotinamide-adenine dinucleotide in the cytoplasm and mitochondria ofrat liver. The Biochemical Journal 103, 514-527 (1967).

aP2 Upregulates Gluconeogenic Gene Expression in a FoxO1-DependentManner

Acting as an adipokine, aP2 directly upregulates expression ofgluconeogenic genes both in liver in vivo, and in hepatocytes in vitro.See, Cao, H., et al. Adipocyte lipid chaperone AP2 is a secretedadipokine regulating hepatic glucose production. Cell metabolism 17,768-778 (2013).

Since the extent of upregulation is modest in cell culture systems,additional experimental settings were examined to obtain more robustupregulation of these genes. It has been discovered that the presence ofsignals mimicking fasting increases the effect of aP2 on geneexpression. For example, forskolin (an activator of adenylate cyclase)and glucagon (data not shown) enhanced the ability of aP2 to upregulateG6pc and Pck1 gene expression in primary hepatocytes (FIGS. 38A and38B). Conversely, blocking cAMP-PKA signaling with H89 abolished theeffect of aP2 (FIGS. 38C and 38D).

Further, the molecular basis of gluconeogenic gene regulation by aP2 inthis setting has been identified as described herein. Both delipidatedaP2 and a mutant form of aP2 that lacks lipid binding ability (R126L,Y128F) (Erbay, E., et al. Reducing endoplasmic reticulum stress througha macrophage lipid chaperone alleviates atherosclerosis. Nature Medicine15, 1383-1391 (2009)) failed to upregulate gluconeogenic gene expressionin the presence of forskolin, (FIGS. 32A and 32B), indicating that aP2lipid cargo is an important mediator of this activity.

siRNA knockdown was used to screen several key hepatic transcriptionfactors and cofactors, such as FoxO1, Hypoxia-inducible factor 1α(Hif1α), Signal transducer and activator of transcription 3 (Stat3),Peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α,Ppargc1a), CCAAT-enhancer-binding proteins α (C/EBPα, Cebpa) andGlucocorticoid Receptor (GR, Nuclear receptor subfamily 3 group C member1, Nr3c1) for their ability to abrogate the effect of aP2 on geneexpression. Among these, knockdown of FoxO1, which has previously beenshown to play a critical role in the transcriptional regulation ofgluconeogenic gene expression (see, Lin, H. V. & Accili, D. Hormonalregulation of hepatic glucose production in health and disease. Cellmetabolism 14, 9-19 (2011)), most markedly blunted the effect of aP2 onG6pc expression (FIGS. 32C, 39A-39H).

This finding was further validated by adenovirus-mediated shRNAdelivery. Again adenovirus-mediated FoxO1 knockdown significantlydiminished the upregulation of G6pc gene expression induced by aP2(FIGS. 32D and 32E). Consistent with this finding, aP2 increased theactivity of a FoxO-responsive luciferase reporter (FHRE) in primaryhepatocytes, and this effect was more pronounced in the presence offorskolin (FIG. 32F). Treatment with aP2 did not influence theexpression levels of Foxo1 (FIG. 39H). Furthermore, aP2 increasedglucose production from hepatocytes in a FoxO1-dependent manner (FIG.32G).

Extracellular aP2 Alters Fatty Acid Metabolism in Hepatocytes

Since aP2 is a fatty acid binding protein and genetic deficiency andantibody-mediated neutralization of aP2 are known to alter systemicfatty acid metabolism (Hotamisligil, G. S., et al. Uncoupling of obesityfrom insulin resistance through a targeted mutation in aP2, theadipocyte fatty acid binding protein. Science (New York, N.Y.) 274,1377-1379 (1996), Maeda, K., et al. Adipocyte/macrophage fatty acidbinding proteins control integrated metabolic responses in obesity anddiabetes. Cell metabolism 1, 107-119 (2005); Cao, H., et al. Regulationof metabolic responses by adipocyte/macrophage Fatty Acid-bindingproteins in leptin-deficient mice. Diabetes 55, 1915-1922 (2006)), theeffect of exogenous aP2 treatment on fatty acid metabolism inhepatocytes was assessed. The effect of aP2 was significantly diminishedin the presence of etomoxir, a fatty acid oxidation inhibitor (FIGS. 33Aand 33B). This data suggests that aP2 is regulating gluconeogenic geneexpression by inhibiting fatty acid oxidation. Consistent with thisfinding, aP2 decreased the palmitate-stimulated increase in fatty acidoxidation, as measured by oxygen consumption rate after palmitateinjection (FIG. 33C). Furthermore, aP2 robustly increased fatty aciduptake into hepatocytes (FIG. 33D) suggesting that the extent ofinhibition of fatty acid oxidation by aP2 observed (FIG. 33C) may be anunderestimation. Moreover, exogenously supplied palmitate increased theextent of gluconeogenic gene expression induced by aP2 (FIGS. 33E and33F).

Once taken up into cells, fatty acids are esterified with CoA to formfatty acyl-CoA thioesters, and transported to organelles such asmitochondria and endoplasmic reticulum where they are utilized foroxidation and lipid synthesis. See, Mashek, D. G. & Coleman, R. A.Cellular fatty acid uptake: the contribution of metabolism. Currentopinion in lipidology 17, 274-278 (2006) and Anderson, C. M. & Stahl, A.SLC27 fatty acid transport proteins. Molecular aspects of medicine 34,516-528 (2013). Following aP2 treatment, an increase of fatty aciduptake with concomitant decline of fatty acid oxidation would presumablycause accumulation of cytosolic fatty acyl-CoAs, which has been shown toplay regulatory roles in metabolic diseases. See, DeFronzo, R. A.Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis:the missing links. The Claude Bernard Lecture 2009. Diabetologia 53,1270-1287 (2010). To test whether the effect of palmitate on aP2-inducedgluconeogenic gene expression involves cytosolic acyl-CoA, the acyl-CoAsynthetase inhibitor, Triacsin C, was used. See, Mashek, D. G. &Coleman, R. A. Cellular fatty acid uptake: the contribution ofmetabolism. Current opinion in lipidology 17, 274-278 (2006). Chemicalinhibition of acyl-CoA synthetase significantly diminished the abilityof exogenous palmitate to enhance the aP2 effect on gene expression(FIGS. 33G and 33H). Furthermore, treating primary hepatocytes with aP2induced a significant increase in the accumulation of nuclear fattyacyl-CoA (FIG. 33I). Taken together, these data indicate that both lipidflux and turnover in liver cells are modulated by aP2, and that thisunderlies the effect of aP2 on gluconeogenic gene expression.

A Novel Repressive Complex, FoxO1/CtBP2, is Modulated by aP2

FoxO1 has not been previously shown to be regulated by the intracellularlipid milieu. Therefore, molecules which might link lipid signaling toFoxO1 activation were examined. C-terminal binding proteins (CtBPs) aretranscriptional repressors that have been shown to bind to not onlynicotinamide adenine dinucleotide (NAD⁺/NADH) but also fatty acyl-CoAs.See, Nardini, M., et al. CtBP/BARS: a dual-function protein involved intranscription co-repression and Golgi membrane fission. The EMBO journal22, 3122-3130 (2003). Importantly, NAD⁺/NADH binding induces aconformational change of CtBPs and promotes homo-dimerization andformation of an active repressor form, while acyl-CoA binding inducesmonomeric conformation. See, Nardini, M., et al. CtBP/BARS: adual-function protein involved in transcription co-repression and Golgimembrane fission. The EMBO journal 22, 3122-3130 (2003). In ourexperimental system, we examined the role of each of the two CtBPisoforms on aP2-mediated gene expression. While knockdown of CtBP1 hadno impact on aP2-induced gene expression, CtBP2 knockdown blocked theeffect of aP2 (See, FIGS. 34A, 34B, 40A, 40B, 40C and 40D). Notably,although Hepatocyte nuclear factor 4 α (HNF4α) is a well-knowngluconeogenic transcription factor with binding affinity against longchain fatty acyl-CoAs (Hertz, R., Magenheim, J., Berman, I. & Bar-Tana,J. Fatty acyl-CoA thioesters are ligands of hepatic nuclearfactor-4alpha. Nature 392, 512-516 (1998)) an effect of HNF4α knockdownon aP2-mediated gene expression was not observed (FIGS. 40E and 40F).

CtBPs bind PxDLx motifs in DNA-binding proteins. See, Chinnadurai, G.Transcriptional regulation by C-terminal binding proteins. Theinternational journal of biochemistry & cell biology 39, 1593-1607(2007) and Turner, J. & Crossley, M. The CtBP family: enigmatic andenzymatic transcriptional co-repressors. BioEssays: news and reviews inmolecular, cellular and developmental biology 23, 683-690 (2001).Sequence analysis revealed PxDLx motifs in multiple mouse and human FoxOproteins (FIG. 34C), suggesting the potential for direct interactionbetween FoxOs and CtBPs. Co-immunoprecipitation experiments revealed anendogenous FoxO1/CtBP complex in primary hepatocytes (FIGS. 34D and34E). Mutation or deletion of the PSDL motif in FoxO1 specificallyreduced the interaction between FoxO1 and CtBP2, but did not diminishinteraction between FoxO1 and CtBP1 or another binding partner,β-catenin (FIGS. 34F, 34G, 41A and 41B), indicating that CtBP2 directlybinds to FoxO1 through its PxDLx motif whereas CtBP1 may bind to FoxO1indirectly or through other interaction site(s). Remarkably, uponstimulation of primary hepatocytes with aP2, FoxO1 dissociated fromCtBP2 (see FIGS. 34H, and 41C).

While the effect of NAD⁺/NADH on transcriptional repression by CtBPs hasbeen thoroughly investigated, [Zhang, Q., Piston, D. W. & Goodman, R. H.Regulation of corepressor function by nuclear NADH. Science (New York,N.Y.) 295, 1895-1897 (2002); Kumar, V., et al. Transcription corepressorCtBP is an NAD(+)-regulated dehydrogenase. Molecular cell 10, 857-869(2002); Fjeld, C. C., Birdsong, W. T. & Goodman, R. H. Differentialbinding of NAD+ and NADH allows the transcriptional corepressorcarboxyl-terminal binding protein to serve as a metabolic sensor.Proceedings of the National Academy of Sciences of the United States ofAmerica 100, 9202-9207 (2003); and Thio, S. S., Bonventre, J. V. & Hsu,S. I. The CtBP2 co-repressor is regulated by NADH-dependent dimerizationand possesses a novel N-terminal repression domain. Nucleic AcidsResearch 32, 1836-1847 (2004)), the effect of fatty acyl-CoA on thetranscriptional repressor activity of CtBPs has not been studied. See,Valente, C., Spano, S., Luini, A. & Corda, D. Purification andfunctional properties of the membrane fissioning protein CtBP3/BARS.Methods In Enzymology 404, 296-316 (2005). To elucidate this, increasingconcentrations of oleoyl-CoA were added into cell lysates and it wasfound that this treatment resulted in dissociation of the FoxO1/CtBP2complexes in a dose-dependent manner (FIG. 34I). In contrast, we alsoobserved increased FoxO1/CtBP2 complex formation in the presence ofexogenous NADH (see FIG. 34J). CtBPs have been reported to be a redoxsensor that senses NAD⁺/NADH ratio (Zhang, Q., Piston, D. W. & Goodman,R. H. Regulation of corepressor function by nuclear NADH. Science (NewYork, N.Y.) 295, 1895-1897 (2002); Chinnadurai, G. Transcriptionalregulation by C-terminal binding proteins. The International Journal OfBiochemistry & Cell Biology 39, 1593-1607 (2007)), thus whetherFoxO1/CtBP2 complex can respond to the cytosolic redox state orNAD⁺/NADH ratio was examined. HEK293 cells transfected with FLAG-FoxO1and CtBP2 were treated with different ratios of extracellularlactate/pyruvate to change the cytosolic redox state. Formation of theFoxO1/CtBP2 complex was enhanced in cells with a high extracellularlactate/pyruvate ratio (low cytosolic NAD⁺/NADH ratio) (see FIG. 34K).These data suggest that the FoxO1/CtBP2 complex is regulated not only byfatty acyl-CoA but also by NAD⁺/NADH. Therefore we next asked whetheraP2 treatment directly alters the cytosolic NAD⁺/NADH ratio. Since thecytosolic NAD⁺/NADH ratio is reflected in the cellular pyruvate/lactateratio assuming equilibrium of lactate dehydrogenase, we measuredcellular lactate/pyruvate ratio in the same experimental setting as inFIGS. 33E and 33F. The lactate/pyruvate ratio tended to be increased byaP2 in the presence or absence of palmitate but the difference was notsignificant (see FIG. 41D). The inhibition of fatty acid oxidation wasreported to increase lactate production. See, Pike, L. S., Smift, A. L.,Croteau, N. J., Ferrick, D. A. & Wu, M. Inhibition of fatty acidoxidation by etomoxir impairs NADPH production and increases reactiveoxygen species resulting in ATP depletion and cell death in humanglioblastoma cells. Biochimica Et Biophysica Acta 1807, 726-734 (2011).Therefore, this trend toward an increase of lactate/pyruvate ratio couldbe secondary to the inhibition of fatty acid oxidation. Here weinvestigated direct action of aP2 in relatively acute experimentalsettings. Thus, aP2 regulates FoxO1/CtBP2 complex formation mainly bymodulating cellular fatty acyl-CoA content although aP2 might influenceNADH/NAD⁺ ratio secondarily in a long term process. FoxO activity isregulated in part by nuclear cytoplasmic shuttling throughposttranslational modifications such as phosphorylation and acetylation.See, Eijkelenboom, A. & Burgering, B. M. FOXOs: signalling integratorsfor homeostasis maintenance. Nature Reviews. Molecular Cell Biology 14,83-97 (2013). It was found that aP2 treatment tended to increase thenuclear content of FoxO1 in hepatocytes, although the change ofcytosolic content was negligible (see FIGS. 34L and 41E). In addition,nuclear cytoplasmic shuttling of CtBP2 by these stimuli were notobserved. Interestingly, aP2 and forskolin treatment did not obviouslyalter the levels of FoxO1 phosphorylation and acetylation (see FIG.41F), modifications, which are known to regulate nuclear-cytoplasmicshuttling of the protein.

The promoter occupancy of CtBP2 as well as FoxO1 by chromatinimmunoprecipitation (ChIP) assay were further investigated. Exposure toaP2 caused dissociation of CtBP2 from the G6pc promoter in the absenceor presence of forskolin (FIGS. 34M and 34N). FoxO1 was recruited toG6pc promoter by aP2 treatment although the effect was marginal (FIGS.34M and 34N).

CtBP2 Tightly Regulates Gluconeogenic Gene Expression

The characteristics of this newly identified transcriptional system werefurther investigated. CtBPs are known to be repressors, but theyfunction as activators in some cases. See, Fang, M., et al.C-terminal-binding protein directly activates and represses Wnttranscriptional targets in Drosophila. The EMBO journal 25, 2735-2745(2006). Therefore, the effects of CtBP2 on FoxO1-mediated gluconeogenicgene expression were verified. CtBP2 knockdown upregulated expression ofG6pc (FIG. 35A) and Pck1 (data not shown) in primary hepatocytes, andthis effect was enhanced in the presence of forskolin, which was alsothe case with aP2-mediated upregulation of these genes. Furthermore, theupregulation of these genes by CtBP2 knockdown was diminished bysimultaneous knockdown of FoxO1 (FIG. 35A). CtBP2 knockdown alsoactivated the FoxO-responsive luciferase reporter (FIG. 35B).Conversely, CtBP2 overexpression downregulated G6pc expression atbaseline and forskolin-induced upregulation of G6pc and Pck1 wererobustly suppressed by CtBP2 overexpression compared to β-glucuronidase(GUS) overexpression (see FIGS. 35C, 35D and 35E). To rule out thepossibility of general transcriptional repression occurring uponoverexpression of CtBP2, the expression of multiple hepatocyte genes wasassessed. Some genes regulating cellular metabolism were upregulated(e.g. Acadm) others were unchanged (e.g. Alb, Creb1) (see FIGS. 36A-36N)and data not shown), suggesting a specific effect of CtBP2.Downregulation of FoxO1 target genes following CtBP2 overexpression wasalso verified by FoxO responsive luciferase reporter activity (FIG.35F).

Whether insulin and cAMP signaling regulate the FoxO1/CtBP2transcriptional complex, since these pathways have been previously shownto regulate FoxO1 transcriptional activity, was also investigated. See,Brunet, A., et al. Akt promotes cell survival by phosphorylating andinhibiting a Forkhead transcription factor. Cell 96, 857-868 (1999);Nakae, J., et al. Regulation of insulin action and pancreatic beta-cellfunction by mutated alleles of the gene encoding forkhead transcriptionfactor Foxo1. Nature Genetics 32, 245-253 (2002); and Mihaylova, M. M.,et al. Class IIa histone deacetylases are hormone-activated regulatorsof FOXO and mammalian glucose homeostasis. Cell 145, 607-621 (2011). Asshown herein, acute activation of these pathways did not robustly alterthe formation of the FoxO1/CtBP2 complex although a mild increase ofFoxO1/CtBP2 complex by insulin treatment was observed (FIG. 35G), whichmay be caused by an increased NADH/NAD⁺ ratio through enhancedglycolytic flux.

Circulating aP2 Activity Dissociates the FoxO1/CtBP2 Holocomplex In Vivo

To further explore the relevance of aP2 modulation of the FoxO1/CtBP2complex, the transcriptional complex formation was analyzed in vivo.Since decreased NAD⁺ and increased fatty acyl-CoA content in the liverof obese mice have been reported (Samuel, V. T., et al. Mechanism ofhepatic insulin resistance in non-alcoholic fatty liver disease. TheJournal of Biological Chemistry 279, 32345-32353 (2004); Hammond, L. E.,et al. Mitochondrial glycerol-3-phosphate acyltransferase-1 is essentialin liver for the metabolism of excess acyl-CoAs. The Journal ofBiological Chemistry 280, 25629-25636 (2005); Cheng, Z., et al. Foxo1integrates insulin signaling with mitochondrial function in the liver.Nature Medicine 15, 1307-1311 (2009); and Eckel-Mahan, K. L., et al.Reprogramming of the circadian clock by nutritional challenge. Cell 155,1464-1478 (2013)) dysfunctional corepressor activity of CtBPs couldunderlie the unregulated hepatic gluconeogenesis in obesity. In theliver of both genetically-induced and diet-induced obese mice, whichhave elevated levels of circulating aP2 (Cao, H., et al. Adipocyte lipidchaperone aP2 is a secreted adipokine regulating hepatic glucoseproduction. Cell Metabolism 17, 768-778 (2013)), FoxO1/CtBP2 associationwas dramatically reduced compared to lean controls (see FIGS. 36A, 36B,36C and 36D). In addition, both genetic deficiency of aP2 andantibody-mediated neutralization of aP2 (using anti-aP2 monoclonal CA33)robustly enhanced formation of the FoxO1/CtBP2 complex (FIGS. 36E, 36F,36G and 36H), consistent with the reduction in hepatic glucoseproduction in these animals. See, Maeda, K., et al. Adipocyte/macrophagefatty acid binding proteins control integrated metabolic responses inobesity and diabetes. Cell Metabolism 1, 107-119 (2005); Cao, H., et al.Adipocyte lipid chaperone aP2 is a secreted adipokine regulating hepaticglucose production. Cell Metabolism 17, 768-778 (2013).

To determine whether elevated circulating aP2 was sufficient to disruptthe FoxO1/CtBP2 complex, recombinant aP2 by IP injection into wild-typelean mice for 5 days was administered, which resulted in a mild increaseof serum aP2 levels (FIG. 36I), similar to the level observed in obesemice [Cao, H., et al. Adipocyte lipid chaperone aP2 is a secretedadipokine regulating hepatic glucose production. Cell Metabolism 17,768-778 (2013)]. This short term increase in circulating aP2 resulted insignificant reduction in FoxO1/CtBP2 interaction in the liver (FIGS. 36Jand 36K), which led to a selective increase in G6pc expression (FIG.36L). Importantly, this acute treatment did not affect body weight orserum metabolic parameters such as insulin, glucagon, free fatty acidsor glycerol (FIGS. 43A, 43B, 43C, 43D, 43E, 43F and 43G).

Since CtBP2 overexpression in hepatocytes robustly downregulatedgluconeogenic gene expression (FIGS. 35D and 35E), the therapeuticpotential of this signaling pathway was investigated in vivo byoverexpressing CtBP2 in the liver of diet-induced obese mice byadenovirus-mediated gene delivery. Three days after adenovirustransduction, CtBP2 overexpression normalized fasting blood glucoselevels in obese mice without inducing weight loss (FIGS. 37A, 37B, and45A). In an independent cohort, 3 days of adenoviral CtBP2overexpression in obese mice resulted in marked improvement in glucosetolerance (FIG. 37C). This improvement was not due to changes in insulinsensitivity, as determined by insulin tolerance test after 5 days ofadenoviral transduction (FIGS. 37D, 45E and 45F). To more directlyassess hepatic glucose production in this model, a pyruvate tolerancetest was performed, and it was observed that CtBP2 overexpressionresulted in a significantly blunted glucose excursion following pyruvateinjection (FIG. 37Q). Consistent with this finding, G6pc expression inliver was robustly downregulated in CtBP2 overexpressing animals whereasAlb gene expression was not affected (FIGS. 37E, 37F and 37G).

Furthermore, a reduction of hepatic lipid accumulation in obese micefollowing 7 days of CtBP2 overexpression (FIG. 37H) was observed.Dramatic improvement of liver steatosis is another feature of geneticdeletion and antibody-mediated neutralization of aP2. See, Maeda, K., etal. Adipocyte/macrophage fatty acid binding proteins control integratedmetabolic responses in obesity and diabetes. Cell Metabolism 1, 107-119(2005); Cao, H., et al. Regulation of metabolic responses byadipocyte/macrophage Fatty Acid-binding proteins in leptin-deficientmice. Diabetes 55, 1915-1922 (2006). The reduction of steatosis by CtBP2overexpression was also verified by the measurement of livertriglycerides (FIG. 37I) and this was accompanied by a reduction ofserum alanine aminotransferase (ALT) levels (FIG. 37J). Consistent withthese findings, lipogenic gene expression was robustly downregulated byCtBP2 overexpression (FIGS. 37K, 37L, 37M, 37N and 37O). This expressionprofile was also observed in primary hepatocyte in a cell autonomousmanner (FIGS. 45A, 45B, 45C and 45D). CtBP2 overexpression decreases theexpression levels of these genes in FoxO1 knockout hepatocytes (FIGS.45E and 45F), which suggests that CtBP2 regulates lipogenic geneexpression in a FoxO1 independent manner. A role for CtBP2 in repressinghepatic steatosis was also shown by CtBP2 loss of function. Fourteendays after transduction of Ad/shCtBP2, CtBP2 knockdown resulted in anaccumulation of triglyceride in the liver in lean mice on a normal chowdiet (FIGS. 45G and 45H), which was accompanied by increased expressionof lipogenic genes (FIGS. 45I and 45J).

In summary, the molecular mechanisms underlying the action of thecirculating aP2 protein were investigated, and a novel transcriptionalholocomplex composed of FoxO1 and CtBP2 was identified which sensescellular nutrients such as fatty acyl-CoA and NAD⁺/NADH. By modulatingfatty acid metabolism, aP2 mediates dissociation of the FoxO1/CtBP2complex, liberating FoxO1 to drive gluconeogenic gene expression (FIG.37P).

Furthermore, the adipokine aP2 is elevated under fasting conditions,where it acts to increase hepatic glucose production through modulationof FOXO1 activity. In the pancreatic beta cell, FOXO1 is a key regulatorof beta cell function, acting as a counter-regulatory transcriptionfactor to PDX1 (pancreatic and duodenal homeobox 1). Under stressconditions, such as prolonged fasting, FOXO1 translocates to the nucleuswhere it displaces PDX1 binding and activates the expression ofstress-response genes including those for antioxidant and unfoldedprotein response (UPR) proteins. Addition of recombinant aP2 to primarymouse and human islets promoted the translocation of FOXO1 (FIG. 46A),suggesting that aP2 may act to modulate beta cell function through aFOXO1-dependent pathway. Further evidence of this is shown withincreased expression of FOXO1 target genes and subsequent proteins after24 hr treatment in INS1 cells (FIG. 46B and FIG. 46C).

In the absence of aP2, FOXO1 translocation is reduced, thus allowing forincreased activity of PDX1. Enhanced PDX1 activity would correspond toincreased insulin expression, enhanced glucose sensing due toup-regulation of glucose transporter and glucokinase, and reducedsusceptibility to apoptosis. Thus, depletion of aP2 through geneticdeletion or antibody treatment, may act to prevent T1D through aFOXO1-dependent mechanism.

Example 7 aP2 Deficiency Results in Improved Airway Function in aP2Knockout Mice Upon Inhalation of a Bronchoconstrictor Agent(Methacholine)

Wild type and aP2 genetically deficient mice raised on regular dietuntil 11-13 weeks of age were sensitized with 100 micrograms ofovalbumin (OVA) injected intraperitoneally (IP) on experiment day 0 and14. The animals were then nebulized with 1% OVA in PBS on days 28, 30,32 and 33. Experimental measurements were collected on day 34. Airwayresistance was measured during challenge using a bronchial constrictor,methacholine, at increasing concentrations. The mice were nebulized withmethacholine for 20 minutes and repetitive measurements were taken for 3minutes for a total of 12 consecutive measurements using the FlexiVentapparatus (Scireq, Scientific Respiratory Equipment, Canada). Resultsshown in FIG. 47 illustrate that ap2-deficient mice have improved airwayfunction during methacholine challenge compared to wild type mice underthe same conditions.

This specification has been described with reference to embodiments ofthe invention. However, one of ordinary skill in the art appreciatesthat various modifications and changes can be made without departingfrom the scope of the invention as set forth in the claims below.Accordingly, the specification is to be regarded in an illustrativerather than a restrictive sense, and all such modifications are intendedto be included within the scope of invention.

What is claimed is:
 1. A humanized anti-aP2 monoclonal antibody, orantigen binding agent, comprising: (a) a light chain variable regioncomprising one, two, or three complementarity determining regions(CDR-Ls) independently selected from an amino acid sequence of a CDR-L1region, an amino acid sequence of a CDR-L2 region, or an amino acidsequence of a CDR-L3 region, wherein the amino acid sequence of theCDR-L1 region is Seq. ID No. 7; wherein the amino acid sequence of theCDR-L2 region is Seq. ID No. 8; and, wherein the amino acid sequence ofthe CDR-L3 region is selected from Seq. ID No. 9, Seq. ID No. 10, Seq.ID No. 11, or Seq. ID No. 12; and (b) a heavy chain variable regioncomprising one, two, or three complementarity determining regions(CDR-Hs) independently selected from an amino acid sequence of a CDR-H1region, an amino acid sequence of a CDR-H2 region, or an amino acidsequence of a CDR-H3 region, wherein the amino acid sequence of theCDR-H1 region is Seq. ID No.14; wherein the amino acid sequence of theCDR-H2 region is Seq. ID No. 16 or Seq. ID No. 17; and, wherein theamino acid sequence of the CDR-H3 region is selected from Seq. ID No. 19or Seq. ID No.
 20. 2. The humanized anti-aP2 monoclonal antibody, orantigen binding agent, of claim 1, wherein the light chain variableregion comprises Seq. ID No.
 7. 3. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 1, wherein the light chainvariable region comprises Seq. ID No.
 8. 4. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 1, wherein thelight chain variable region comprises Seq. ID No.
 9. 5. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 1,wherein the light chain variable region comprises Seq. ID No.
 10. 6. Thehumanized anti-aP2 monoclonal antibody, or antigen binding agent, ofclaim 1, wherein the light chain variable region comprises Seq. ID No.11.
 7. The humanized anti-aP2 monoclonal antibody, or antigen bindingagent, of claim 1, wherein the light chain variable region comprisesSeq. ID No.
 12. 8. The humanized anti-aP2 monoclonal antibody, orantigen binding agent, of claim 1, wherein the light chain variableregion comprises three complementarity determining regions comprising aCDR-L1 region, a CDR-L2 region, and a CDR-L3 region.
 9. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 8,wherein the CDR-L3 region is Seq. ID No.
 9. 10. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 8, wherein theCDR-L3 region is Seq. ID No.
 10. 11. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 8, wherein the CDR-L3region is Seq. ID No.
 11. 12. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 8, wherein the CDR-L3region is Seq. ID No.
 12. 13. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 1, wherein the heavy chainvariable region comprises Seq. ID No.
 14. 14. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 1, wherein theheavy chain variable region comprises Seq. ID No.
 16. 15. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 1,wherein the heavy chain variable region comprises Seq. ID No.
 17. 16.The humanized anti-aP2 monoclonal antibody, or antigen binding agent, ofclaim 1, wherein the heavy chain variable region comprises Seq. ID No.19.
 17. The humanized anti-aP2 monoclonal antibody, or antigen bindingagent, of claim 1, wherein the heavy chain variable region comprisesSeq. ID No.
 20. 18. The humanized anti-aP2 monoclonal antibody, orantigen binding agent, of claim 1, wherein the heavy chain variableregion comprises three complementarity determining regions comprising aCDR-H1 region, a CDR-H2 region, and a CDR-H3 region.
 19. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 18,wherein the CDR-H2 region is Seq. ID No.
 16. 20. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 18, wherein theCDR-H2 region is Seq. ID No.
 17. 21. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H3region is Seq. ID No.
 19. 22. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H3region is Seq. ID No.
 20. 23. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H1region is Seq. ID No. 14, the CDR-H2 region is Seq. ID No. 16, and theCDR-H3 region is Seq. ID No.
 19. 24. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H1region is Seq. ID No. 14, the CDR-H2 region is Seq. ID No. 17, and theCDR-H3 region is Seq. ID No.
 19. 25. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H1region is Seq. ID No. 14, the CDR-H2 region is Seq. ID No. 16, and theCDR-H3 region is Seq. ID No.
 20. 26. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 18, wherein the CDR-H1region is Seq. ID No. 14, the CDR-H2 region is Seq. ID No. 17, and theCDR-H3 region is Seq. ID No.
 20. 27. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 1, wherein the light chainvariable region comprises an amino acid sequence selected from Seq. IDNo. 446, Seq. ID No. 448, Seq. ID No. 487, Seq. ID No. 488, Seq. ID No.450, or Seq. ID No.
 452. 28. The humanized anti-aP2 monoclonal antibody,or antigen binding agent, of claim 1, wherein the heavy chain variableregion comprises an amino acid sequence selected from Seq. ID No. 455,Seq. ID No. 459, Seq. ID No. 457, Seq. ID No. 461, or Seq. ID No. 463.29. The humanized anti-aP2 monoclonal antibody, or antigen bindingagent, of claim 1, wherein the light chain variable region comprisesSeq. ID No. 446 and the heavy chain variable region is Seq. ID No. 459.30. The anti-aP2 antibody, or antigen binding agent, of claim 1, whereinthe antibody, or antigen binding agent thereof, has a KD for human aP2of about ≧10⁻⁷ M.
 31. A humanized anti-aP2 monoclonal antibody, orantigen binding agent, comprising a light chain variable regioncomprising one, two, or three complementarity determining regions(CDR-Ls) independently selected from an amino acid sequence of a CDR-L1region, an amino acid sequence of a CDR-L2 region, or an amino acidsequence of a CDR-L3 region, wherein the amino acid sequence of theCDR-L1 region is Seq. ID No. 7; wherein the amino acid sequence of theCDR-L2 region is Seq. ID No. 8; and, wherein the amino acid sequence ofthe CDR-L3 region is selected from Seq. ID No. 9, Seq. ID No. 10, Seq.ID No. 11, or Seq. ID No.
 12. 32. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 31, wherein the light chainvariable region comprises Seq. ID No.
 7. 33. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 31, wherein thelight chain variable region comprises Seq. ID No.
 8. 34. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 31,wherein the light chain variable region comprises Seq. ID No.
 9. 35. Thehumanized anti-aP2 monoclonal antibody, or antigen binding agent, ofclaim 31, wherein the light chain variable region comprises Seq. ID No.10.
 36. The humanized anti-aP2 monoclonal antibody, or antigen bindingagent, of claim 31, wherein the light chain variable region comprisesSeq. ID No.
 11. 37. The humanized anti-aP2 monoclonal antibody, orantigen binding agent, of claim 31, wherein the light chain variableregion comprises Seq. ID No.
 12. 38. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 31, wherein the light chainvariable region comprises three complementarity determining regionscomprising a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region. 39.The human anti-aP2 monoclonal antibody, or antigen binding agent, ofclaim 38, wherein the CDR-L3 region is Seq. ID No.
 9. 40. The humananti-aP2 monoclonal antibody, or antigen binding agent, of claim 38,wherein the CDR-L3 region is Seq. ID No.
 10. 41. The human anti-aP2monoclonal antibody, or antigen binding agent, of claim 38, wherein theCDR-L3 region is Seq. ID No.
 11. 42. The human anti-aP2 monoclonalantibody, or antigen binding agent, of claim 38, wherein the CDR-L3region is Seq. ID No.
 12. 43. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 31, wherein the light chainvariable region comprises an amino acid selected from Seq. ID No. 446,Seq. ID No. 448, Seq. ID No. Seq. ID No. 487, Seq. ID No. 488, Seq. IDNo. 450, Seq. ID No.
 452. 44. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 43, wherein the light chainvariable region comprises Seq. ID No.
 448. 45. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 31, furthercomprising a heavy chain variable region.
 46. The anti-aP2 antibody, orantigen binding agent, of claim 31, wherein the antibody, or antigenbinding agent thereof, has a KD for human aP2 of about ≧10⁻⁷ M.
 47. Ahumanized anti-aP2 monoclonal antibody, or antigen binding agent,comprising a heavy chain variable region comprising one, two, or threecomplementarity determining regions (CDR-Hs) independently selected froman amino acid sequence of a CDR-H1 region, an amino acid sequence of aCDR-H2 region, or an amino acid sequence of a CDR-H3 region, wherein theamino acid sequence of the CDR-H1 region is Seq. ID No.14; wherein theamino acid sequence of the CDR-H2 region is Seq. ID No. 16 or Seq. IDNo. 17; and, wherein the amino acid sequence of the CDR-H3 region isselected from Seq. ID No. 19 or Seq. ID No.
 20. 48. The humanizedanti-aP2 monoclonal antibody, or antigen binding agent, of claim 47,wherein the heavy chain variable region comprises Seq. ID No.
 14. 49.The humanized anti-aP2 monoclonal antibody, or antigen binding agent, ofclaim 47, wherein the heavy chain variable region comprises Seq. ID No.16.
 50. The humanized anti-aP2 monoclonal antibody, or antigen bindingagent, of claim 47, wherein the heavy chain variable region comprisesSeq. ID No.
 17. 51. The humanized anti-aP2 monoclonal antibody, orantigen binding agent, of claim 47, wherein the heavy chain variableregion comprises Seq. ID No.
 19. 52. The humanized anti-aP2 monoclonalantibody, or antigen binding agent, of claim 47, wherein the heavy chainvariable region comprises Seq. ID No.
 20. 53. The humanized anti-aP2monoclonal antibody, or antigen binding agent, of claim 47, wherein theheavy chain variable region comprises three complementarity determiningregions comprising a CDR-H1 region, a CDR-H2 region, and a CDR-H3region.
 54. The humanized anti-aP2 monoclonal antibody, or antigenbinding agent, of claim 53, wherein the CDR-H1 region is Seq. ID No. 14,the CDR-H2 region is Seq. ID No. 16, and the CDR-H3 region is Seq. IDNo.
 19. 55. The humanized anti-aP2 monoclonal antibody, or antigenbinding agent, of claim 53, wherein the CDR-H1 region is Seq. ID No. 14,the CDR-H2 region is Seq. ID No. 17, and the CDR-H3 region is Seq. IDNo.
 19. 56. The humanized anti-aP2 monoclonal antibody, or antigenbinding agent, of claim 53, wherein the CDR-H1 region is Seq. ID No. 14,the CDR-H2 region is Seq. ID No. 16, and the CDR-H3 region is Seq. IDNo.
 20. 57. The humanized anti-aP2 monoclonal antibody, or antigenbinding agent, of claim 53, wherein the CDR-H1 region is Seq. ID No. 14,the CDR-H2 region is Seq. ID No. 17, and the CDR-H3 region is Seq. IDNo.
 20. 58. The anti-aP2 antibody, or antigen binding agent, of claim 1,wherein the antibody, or antigen binding agent thereof, has a KD forhuman aP2 of about ≧10⁻⁷ M.
 59. A method of attenuating an aP2-mediateddisorder in a human comprising administering an effective amount of ahumanized anti-aP2 monoclonal antibody or antigen binding agentcomprising: (a) a light chain variable region comprising one, two, orthree complementarity determining regions (CDR-Ls) independentlyselected from an amino acid sequence of a CDR-L1 region, an amino acidsequence of a CDR-L2 region, or an amino acid sequence of a CDR-L3region, wherein the amino acid sequence of the CDR-L1 region is Seq. IDNo. 7; wherein the amino acid sequence of the CDR-L2 region is Seq. IDNo. 8; and, wherein the amino acid sequence of the CDR-L3 region isselected from Seq. ID No. 9, Seq. ID No. 10, Seq. ID No. 11, or Seq. IDNo. 12; and (b) a heavy chain variable region comprising one, two, orthree complementarity determining regions (CDR-Hs) independentlyselected from an amino acid sequence of a CDR-H1 region, an amino acidsequence of a CDR-H2 region, or an amino acid sequence of a CDR-H3region, wherein the amino acid sequence of the CDR-H1 region is Seq. IDNo.14; wherein the amino acid sequence of the CDR-H2 region is Seq. IDNo. 16 or Seq. ID No. 17; and, wherein the amino acid sequence of theCDR-H3 region is selected from Seq. ID No. 19 or Seq. ID No.
 20. 60. Themethod of claim 59, wherein the aP2 mediated disorder is a metabolicdisorder.
 61. The method of claim 60, wherein the metabolic disorder istype I diabetes.
 62. The method of claim 60, wherein the metabolicdisorder is type II diabetes.
 63. The method of claim 60, wherein themetabolic disorder is hyperglycemia.
 64. The method of claim 60, whereinthe metabolic disorder is obesity.
 65. The method of claim 60, whereinthe metabolic disorder is fatty liver disease.
 66. The method of claim60, wherein the metabolic disorder is dyslipidemia.
 67. The method ofclaim 60, wherein the metabolic disorder is polycystic ovary syndrome(POS).
 68. The method of claim 59, wherein the aP2-mediated disorder isa cardiovascular disorder.
 69. The method of claim 68, wherein thecardiovascular disorder is atherosclerosis.
 70. The method of claim 68,wherein the cardiovascular disorder is associated with peri- orpost-menopause.
 71. The method of claim 59, wherein the aP2-mediateddisorder is an inflammation disorder.
 72. The method of claim 71,wherein the inflammation disorder is asthma.
 73. The method of claim 59,wherein the aP2-mediated disorder is a neoplastic disorder.
 74. Themethod of claim 73, wherein the neoplastic disorder is selected fromliposarcoma, mesentery mixed-mullerian tumor, spinal schwannoma,gallbladder cancer, prostate cancer, brain astrocytoma, lung cancer,ovarian cancer, bladder cancer, colon cancer, esophageal cancer,post-menopausal breast cancer, endometrial cancer, kidney cancer, livercancer, or pancreatic cancer.